Substrates 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 a substrate for single molecule organization.

Cross-reference to related applications

Background Art

[0002] The current state of personalized healthcare is predominantly genomics-centered, mainly focusing on quantifying the genes present within an individual. While such techniques have proven to be extremely powerful, they do not provide a complete picture of an individual's health status to a clinician. This is because genes are merely the "blueprints" of an individual and only inform the likelihood of developing a disease. Within an individual, these "blueprints" need to be first transcribed into RNA and then converted into various protein molecules, which are the actual "actors" within the cell, in order to have any effect on the individual's health status.

[0003] Protein concentrations, protein-protein interactions (protein-protein interactions or PPIs), and interactions between proteins and small molecules are intricately linked to the health status of different organs, homeostatic regulatory mechanisms, and the interaction of these systems with the external environment. Therefore, quantitative information on proteins and PPIs is crucial for generating a comprehensive picture of an individual's health status at a given point in time and for predicting any health problems that may arise. For example, the amount of stress on the myocardium (e.g., during a heart attack) can be estimated by measuring the concentrations of troponin I / II and myosin light strands 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), specific cancers (e.g., colorectal cancer or prostate cancer), and infectious diseases (e.g., HIV and Zika). These protein-protein interactions are also essential and increasingly in-demand data sets for drug development. The ability to detect and quantify proteins and other molecules within a given sample of body fluids is an essential component of such healthcare developments. [Overview of the Initiative] [Means for solving the problem]

[0004] The disclosures of this invention generally relate to systems, structures, and methods for the detection and quantification of sample molecules in a sample.

[0005] Provided herein is a method for detecting sample molecules present in a sample, in some embodiments, the method comprising: 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 binding sites from among the plurality of binding sites on the solid support, the supramolecular structure comprising: 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, the supramolecular structure being in an unstable state such that the detector molecule is released from the core structure through the severing of the link between them at a second location.

[0006] Provided herein is a method for detecting sample molecules using a substrate, in some embodiments, the method comprising the steps of: providing a substrate having a plurality of binding sites, wherein each of the plurality of binding sites is associated with a supramolecular structure among a plurality of supramolecular structures, the supramolecular structure comprising a core structure having 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 configured such that the detector molecule is released from the core structure through the severance of the link between them at a second location; and providing a sample so that the supramolecular structure shifts from the unstable state to a stable state. The method comprises the steps of bringing a supramolecular structure into contact with a supramolecular structure, wherein a detector molecule and a capture molecule are linked to each other by binding to a sample molecule, thereby forming a link between the detector molecule and the capture molecule, and each unstable supramolecular structure comprises a detector molecule and a capture molecule separated by a predetermined distance; a step of giving a trigger to break the link between the detector molecule and the core structure at a second location, wherein the detector molecule remains linked to the core structure through linkage with the capture molecule and so that the sample molecule is associated with individual binding sites; and a step of detecting the sample molecule based on a signal provided by the supramolecular structure shifted to a stable state. In other embodiments, the supramolecular structure does not include an integrated detector molecule, and detection is performed via separate steps.

[0007] Provided herein is a method for forming a substrate for detecting sample molecules in a sample, in some embodiments. The method comprises the steps of 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 of the plurality of binding sites, wherein the supramolecular structure comprises a core structure having 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, wherein the supramolecular structure is in an unstable state such that the detector molecule is released from the core structure through the severance of the link between them at a second location, and each separated capture molecule and detector molecule is at a predetermined distance in the unstable state, and in the stable state the detector molecule and capture molecule are linked to each other by binding to the sample 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 so that the sample molecule is associated with individual binding sites.

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

[0009] In some embodiments, for any of the methods disclosed herein, the sample comprises a composite biological sample, and the method provides single-molecule sensitivity, thereby increasing the dynamic range and quantitative capture of various molecular concentrations within the composite biological sample. In some embodiments, for any of the methods disclosed herein, the sample molecule comprises a protein, peptide, peptide fragment, lipid, DNA, RNA, organic molecule, inorganic molecule, complex thereof, or any combination thereof. In some embodiments, for any of the methods disclosed herein, each supramolecular structure is a nanostructure.

[0010] In some embodiments, for any method disclosed herein, each core structure is a nanostructure. In some embodiments, for any method disclosed herein, the multiple core molecules for each core structure are arranged in a predetermined shape and / or have a specified molecular weight. In some embodiments, the predetermined shape is configured to limit or prevent cross-reactivity with other supramolecular structures. In some embodiments, for any method disclosed herein, the multiple core molecules for each core structure comprise one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or a combination thereof. In some embodiments, for any method disclosed herein, each core structure independently comprises a scaffold deoxyribonucleic acid (DNA) origami, a scaffold ribonucleic acid (RNA) origami, a scaffold hybrid DNA:RNA origami, a single-strand DNA tile structure, a multi-strand DNA tile structure, a single-strand RNA origami, a multi-strand RNA tile structure, a hierarchical constituent DNA or RNA origami having multiple scaffolds, a peptide structure, or a combination thereof.

[0011] In some embodiments, for any method disclosed herein, the trigger comprises a deconstruction molecule, a trigger signal, or a combination thereof. In some embodiments, the deconstruction molecule comprises DNA, RNA, peptides, small organic molecules, 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 of the methods disclosed herein, each sample molecule is 1) chemically bonded to a capture molecule of each supramolecular structure, and / or 2) chemically bonded to a detector molecule of each supramolecular structure. In some embodiments, for any of the methods disclosed herein, the capture molecule and detector molecule for each supramolecular structure independently comprise a protein, peptide, antibody, aptamer (RNA and DNA), fluorescent dye molecule, darpin, catalyst, polymerization initiator, polymer such as PEG, or a combination thereof. In some embodiments, with respect to each supramolecular structure, for any method disclosed herein, a) a capture molecule is linked to a core structure through a capture barcode, the capture barcode comprises a first capture linker, a second capture linker, and a capture bridge positioned between the first and second capture linkers, the first capture linker being coupled to a first core linker coupled to a first location on the core structure, and the capture molecule and the second capture linker are linked to each other by coupling to a third capture linker; b) a detector molecule is linked to a core structure through a detector barcode, the detector barcode comprises a first detector linker, a second detector linker, and a detector bridge positioned between the first and second detector linkers, the first detector linker being coupled to a second core linker coupled to a second location on the core structure, and the detector molecule and the second detector linker are linked to each other by coupling to a 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 nucleic acid (DNA or RNA) or a polymer such as PEG of a specific sequence. 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 reaction molecule or a DNA sequence domain.In some embodiments, each reaction molecule independently comprises one or more polymers such as amines, thiols, DBCO, maleimide, biotin, azides, acridites, NHS esters, single-strand nucleic acids (RNA or DNA) of a specific sequence, PEG, or polymerization initiators, or a combination thereof. In some embodiments, the link between the capture barcode and 1) a first core linker and / or 2) a third capture linker comprises a chemical bond. In some embodiments, this chemical bond comprises a covalent bond. In some embodiments, the link between the detector barcode and 1) a second core linker and / or 2) a third detector linker comprises a chemical bond. In some embodiments, this chemical bond comprises a covalent bond. In some embodiments, the trigger cleaves the linkage between 1) a first detector linker and a second core linker and / or 2) a first capture linker and a first core linker. In some embodiments, for any method disclosed herein, the capture molecule is chemically bonded to a third capture linker and / or the detector molecule is chemically bonded to a third detector linker. In some embodiments, the capture molecule is covalently bonded to a third capture linker and / or the detector molecule is covalently bonded to a third detector linker.

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

[0014] In some embodiments, for any method disclosed herein, each supramolecular structure further comprises an anchor molecule linked to a core structure. In some embodiments, the anchor molecule is linked to the core structure through an anchor barcode, the anchor barcode comprising a first anchor linker, a second anchor linker, and an anchor bridge positioned between the first and second anchor linkers, the first anchor linker being coupled to a third core linker coupled to a third location on the core structure, and the anchor molecule being linked to the second anchor linker. In some embodiments, the anchor molecule comprises one or more polymers such as amines, thiols, DBCOs, maleimides, biotins, azides, acridides, NHS esters, single-strand nucleic acids (RNA or DNA) of a specific sequence, PEG, or polymerization initiators, or a combination thereof. In some embodiments, the anchor bridge comprises a polymer core. In some embodiments, the polymer core of the anchor bridge comprises a polymer such as nucleic acids (DNA or RNA) of a specific sequence 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 reaction molecule or a DNA sequence domain. In some embodiments, each anchor reaction molecule independently comprises an amine, thiol, DBCO, maleimide, biotin, azide, acridite, NHS ester, single-strand nucleic acid (RNA or DNA) of a specific sequence, PEG, or one or more polymers such as polymerization initiators, or a combination thereof. In some embodiments, the anchor molecule is linked to the second anchor linker through a chemical bond. In some embodiments, the anchor molecule is covalently bonded 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 a combination thereof. In some embodiments, the first and second locations are located on the first side of the core structure, and the third location is located on the second side of the core structure.

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

[0016] In some embodiments, for any method comprising the step of using multiple supramolecular structures disclosed herein, each core structure of the multiple supramolecular structures is identical to one another. In some embodiments, each supramolecular structure has a specified shape, size, molecular weight, or combination thereof that reduces or eliminates cross-reactions between the multiple supramolecular structures. In some embodiments, each supramolecular structure comprises multiple capture molecules and detector molecules. In some embodiments, each supramolecular structure comprises capture molecules and detector molecules in a specified stoichiometric ratio that reduces or eliminates cross-reactions between the multiple supramolecular structures.

[0017] In some embodiments, for any method comprising the step of using a plurality of supramolecular structures disclosed herein, the instability state with respect to each supramolecular structure further comprises a predetermined distance between the capture molecules and detector molecules such that the occurrence of cross-reactions 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 is reduced or suppressed. In some embodiments, the predetermined distance is from 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. 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. In some embodiments, the plurality of supramolecular structures are mounted on one or more solid substrates. In some embodiments, each of the one or more solid substrates comprises a planar substrate and / or a planar substrate having a patterned or molded surface having an array of bonding sites. In some embodiments, multiple supramolecular structures are arranged on a substrate, the substrate having multiple binding sites, each binding site being associated with a corresponding supramolecular structure. In some embodiments, each binding site in the substrate's binding site array is associated with a maximum of one supramolecular structure, for example, only one supramolecular structure per binding site. In some embodiments, multiple supramolecular structures dispersed on a substrate are configured to detect the same sample molecule. In some embodiments, multiple signaling elements are configured to link to a detector molecule of at least one supramolecular structure that has shifted to a stable state. In some embodiments, each signaling element comprises a fluorescent molecule or microbeads, a fluorescent polymer, a highly charged nanoparticle, or a highly charged polymer. In some embodiments, at least one of the multiple supramolecular structures on the substrate is configured to detect a different sample molecule than at least one of the other supramolecular structures. In some embodiments, individual supramolecular structures are barcoded with a unique barcode that distinguishes each supramolecular structure on the substrate in order to identify the location of each supramolecular structure on the substrate.In some embodiments, multiple signaling elements are linked to a detector molecule of at least one supramolecular structure that has been shifted to a stable state. In some embodiments, each signaling element comprises a fluorescent molecule or microbeads, a fluorescent polymer, a highly charged nanoparticle, or a highly charged polymer.

[0018] Provided herein is a method for forming a substrate, comprising the steps of: providing a base layer; providing a binding layer on the base layer; depositing a top layer on the binding layer; patterning the top layer so as to expose portions of the binding layer corresponding to a plurality of binding sites on the binding layer; and associating molecules or structures at each binding site. In some embodiments, the method includes the step of providing a supramolecular structure associated with each of the plurality of binding sites, wherein the supramolecular structure comprises 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 sample. The techniques of the present invention can generally be used to organize single molecules, for example, one or more core structures of supramolecular structures, i.e., they can find applications in a variety of different fields. In embodiments, the techniques of the present invention can generally be used to organize single quantum dots. In some embodiments, for any of the methods disclosed herein, an additional step may be performed to regenerate the formed substrate after sample detection is complete. Regeneration may include the step of removing all or part of the supramolecular structure.

[0019] In some embodiments, a specimen or specimen molecule within the sample is detected. The specimen may comprise biological particles or biomolecules. In some embodiments, the specimen molecule comprises chemical compounds, proteins, peptides, peptide fragments, lipids, nucleic acids, DNA, RNA, organic molecules, viral particles, exosomes, organelles, or complexes thereof. In some embodiments, the sample comprises tissue biopsy material, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial and / or viral samples, or fungal tissue, or a combination thereof.

[0020] Provided herein, in some embodiments, is a substrate for detecting one or more sample molecules in a sample, the substrate comprising a plurality of binding sites associated with each 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 released from the core structure through the severance of the link between them at a second location, and each supramolecular structure being configured to shift from the unstable state to a stable state through interaction between the detector molecule, the capture molecule, and each of the one or more sample molecules, and upon interaction with a trigger, each supramolecular structure shifted to the stable state provides a signal for detecting each sample molecule.

[0021] Provided herein, in some embodiments, is a substrate for detecting one or more sample molecules in a sample, the substrate comprising a base layer, a binding layer on the base layer, a patterned upper 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 of the plurality of binding sites. The supramolecular structure comprises a core structure having 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 released from the core structure through the severance of the link between them at a second location, and each supramolecular structure is configured to shift from the unstable state to a stable state through interactions between the detector molecule, the capture molecule, and each of the one or more sample molecules.

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

[0023] In some embodiments, the core structures of each of the multiple supramolecular structures are identical to one another. In some embodiments, each supramolecular structure has a specified shape, size, molecular weight, or combination thereof that reduces or eliminates cross-reactions between the multiple supramolecular structures. In some embodiments, each supramolecular structure comprises multiple capture molecules and detector molecules. In some embodiments, each supramolecular structure comprises capture molecules and detector molecules in a specified stoichiometric ratio that reduces or eliminates cross-reactions between the multiple supramolecular structures. In some embodiments, the unstable state with respect to each supramolecular structure further comprises capture molecules and detector molecules separated by a predetermined distance that reduces or suppresses the occurrence of cross-reactions 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 from 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 captured molecule and the detector molecule of each supramolecular structure. In some embodiments, the solid substrate comprises a planar substrate. In some embodiments, multiple supramolecular structures are arranged on a substrate, and the substrate comprises multiple binding sites, each binding site configured to link with a corresponding supramolecular structure. In some embodiments, multiple supramolecular structures are configured to detect the same sample molecule. In some embodiments, multiple signaling elements are configured to link with the detector molecule of at least one supramolecular structure that has shifted to a stable state. In some embodiments, each signaling element comprises a fluorescent molecule or microbeads, a fluorescent polymer, a highly charged nanoparticle, or a highly charged polymer. In some embodiments, at least one of the multiple supramolecular structures is configured to detect a different sample molecule than the other supramolecular structures.

[0025] In some embodiments, the supramolecular structure has a specified shape, size, molecular weight, or combination thereof, such that it reduces or eliminates cross-reactions with other supramolecular structures and / or aligns 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 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 supramolecular structure can be sized and shaped such that two supramolecular structures are not considered to fit on a single binding site. In some embodiments, the supramolecular structure can be smaller than the individual binding site in at least one dimension when associated with it. In some embodiments, the supramolecular structure comprises multiple capture and detector molecules. In some embodiments, the supramolecular structure comprises capture molecules and detector molecules in a specified stoichiometric ratio such that it reduces or eliminates cross-reactions with other supramolecular structures.

[0026] Here, specific embodiments of the disclosed devices, delivery systems, or methods of the present invention will be described below with reference to the drawings. None of this detailed description is intended to suggest that any particular component, feature, or step is essential to the present invention.

Brief Description of the Drawings

[0027] [Figure 1A] A diagram depicting an exemplary supramolecular structure and related subordinate components. [Figure 1B] A diagram depicting an exemplary supramolecular structure and related subordinate components. [Figure 2] A diagram depicting an exemplary three-arm nucleic acid junction-based assembled supramolecular structure and related subordinate components. [Figure 3] A diagram depicting exemplary individual subordinate components of the three-arm nucleic acid junction-based supramolecular structure of FIG. 2. [Figure 4] A diagram depicting an exemplary deconstructing molecule corresponding to the subordinate components of the three-arm nucleic acid junction-based supramolecular structure of FIG. 2. [Figure 5] [[ID=,23]]A diagram depicting an exemplary DNA origami-based assembled supramolecular structure and related subordinate components. [Figure 6] A diagram depicting exemplary individual subordinate components of the DNA origami-based supramolecular structure of FIG. 5. [Figure 7] A diagram depicting an exemplary deconstructing molecule corresponding to the subordinate components of the DNA origami-based supramolecular structure of FIG. 5. [Figure 8] An exemplary diagram of a supramolecular structure in an unstable state before and after receiving a trigger (e.g., interaction with a deconstructing molecule). [Figure 9] An exemplary diagram of a supramolecular structure in a stable state before and after receiving a trigger (e.g., interaction with a deconstructing molecule). [Figure 10] An exemplary diagram of a supramolecular structure that shifts from an unstable state to a stable state after interaction with an analyte molecule, and of each configuration before and after receiving a trigger (e.g., interaction with a deconstructing molecule). [Figure 11] This diagram illustrates a supramolecular structure that shifts from a stable state to an unstable state after interacting with a sample molecule, as well as the respective configurations before and after receiving a trigger (e.g., interaction with a deconstruction molecule). [Figure 12] This is an illustrative diagram of a method for detecting and quantifying sample molecules using multiple supramolecular structures. [Figure 13] This is an illustrative diagram of a method for detecting and quantifying sample molecules using multiple supramolecular structures mounted on a planar substrate. [Figure 14] This is an illustrative diagram of a technique for forming an array of binding sites for supramolecular structures, including DNA origami, on a substrate. [Figure 15] This is an illustrative diagram of a technique for forming an array of binding sites for supramolecular structures on a substrate. [Figure 16] This is an illustrative diagram of a technique for forming an array of binding sites for supramolecular structures on a substrate. [Figure 17] This is an illustrative diagram of a technique for forming an array of binding sites for supramolecular structures on a substrate. [Figure 18] This is an illustrative diagram of a substrate coupled to a detection system. [Figure 19] This is an illustrative diagram of a substrate coupled to a field-effect transistor detection system. [Figure 20] This is an illustrative diagram of a substrate coupled to an optical detection system. [Figure 21] This is an illustrative diagram of the workflow for refurbishing circuit boards. [Figure 22] This is an illustrative diagram of a technique for forming an array of binding sites for supramolecular structures, including DNA origami, on a substrate. [Figure 23] This is an illustrative diagram of a technique for forming an array of binding sites for supramolecular structures, including DNA origami, on a substrate. [Figure 24] This is an illustrative diagram of a technique for forming an array of binding sites for supramolecular structures, including DNA origami, on a substrate. [Modes for carrying out the invention]

[0028] Provided herein are structures and methods for detecting one or more sample molecules present in a sample. In some embodiments, one or more sample molecules are detected using one or more supramolecular structures bound to a substrate, such as a solid support. The solid support may include multiple binding sites that accept the supramolecular structures at individual binding sites. Thus, the substrate allows for a single-molecule organization for the supramolecular structures. In one example, each of the multiple binding sites is associated with a single supramolecular structure. The sample can be detected at one or more of the multiple binding sites. Desired analytical functions can be generated based on the patterning and arrangement of the supramolecular structures on the substrate, and the properties of each supramolecular structure, particularly its constituent elements.

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

[0030] sample In some embodiments, the sample comprises an aqueous solution containing proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the sample molecules in the sample comprise proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the sample molecules comprise intact proteins, denatured proteins, partially or completely degraded proteins, peptide fragments, denatured nucleic acids, degraded nucleic acid fragments, complexes thereof, or combinations thereof. In some embodiments, the sample is obtained from tissues, cells, tissue and / or cellular environments, or combinations thereof. In some embodiments, the sample comprises tissue biopsy material, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial samples, viral samples, fungal tissues, or combinations thereof. In some embodiments, the sample is isolated by or without purification from a primary source such as cells, tissues, body fluids (e.g., blood), environmental samples, or combinations thereof. In some embodiments, cells are lysed using mechanical processing or other cell lysis techniques (e.g., lysis buffer). In some embodiments, samples are filtered using mechanical processing (e.g., centrifugation), microfiltration, chromatographic column, other filtration methods, or a combination thereof. In some embodiments, samples are treated with one or more enzymes to isolate one or more nucleic acids or one or more proteins. In some embodiments, samples comprise intact proteins, denatured proteins, partially or completely degraded proteins, peptide fragments, denatured nucleic acids, or degraded nucleic acid fragments. In some embodiments, samples are collected from one or more individuals, one or more animals, one or more plants, or a combination thereof. In some embodiments, samples are collected from individuals, animals, and / or plants with diseases or disorders comprising infectious diseases, immunodeficiencies, cancer, genetic disorders, degenerative diseases, lifestyle-related diseases, injuries, rare diseases, age-related diseases, or a combination thereof.

[0031] supramolecular structure In some embodiments, the supramolecular structure is a programmable structure capable of spatially organizing molecules. In some embodiments, the supramolecular structure comprises a plurality of molecules linked to one another. In some embodiments, the plurality of molecules of the supramolecular structure interact with at least some of each other. In some embodiments, the supramolecular structure has a specific shape. In some embodiments, this supramolecular nanostructure has a specified molecular weight based on its plurality of molecules. In some embodiments, the supramolecular structure is a nanostructure. In some embodiments, the plurality of molecules are linked to one another by bonding, chemical bonding, physical attachment, or a combination thereof. In some embodiments, the supramolecular structure comprises molecular entities of a specific shape and molecular weight formed from a distinct number of smaller molecules that interact with each other in particular. In some embodiments, the structural, chemical, and physical properties of the supramolecular structure are explicitly designed. In some embodiments, the supramolecular structure comprises a plurality of dependent components 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] Figures 1A and 1B provide exemplary embodiments of a supramolecular structure 40 comprising a core structure 13 and capture molecules 2. As shown in Figure 1A, the supramolecular structure 40 may, in embodiments, comprise 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 to one another. 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 singular molecules linked to one another. In some embodiments, the one or more core molecules comprise about 2 to about 1000 singular molecules. In some embodiments, the one or more core molecules interact with each other to determine a particular shape of the supramolecular structure. In some embodiments, the multiple core molecules interact with each other through reversible non-covalent interactions. In some embodiments, the particular shape of the core structure is a three-dimensional (3D) configuration. In some embodiments, the one or more molecules give a particular molecular weight. In some embodiments, the core structure 13 is a nanostructure. In some cases, one or more core molecules comprise one or more nucleic acid strands (e.g., DNA, RNA, non-natural nucleic acids), one or more branched nucleic acids, one or more peptides, one or more small molecules, or a combination 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 scaffold deoxyribonucleic acid (DNA) origami, scaffold ribonucleic acid (RNA) origami, scaffold hybrid DNA:RNA origami, single-strand DNA tile structure, multi-strand DNA tile structure, single-strand RNA origami, multi-strand RNA tile structure, hierarchical constituent DNA or RNA origami with multiple scaffolds, peptide structures, or a combination thereof. In some embodiments, DNA origami is scaffolded. In some embodiments, RNA origami is scaffolded. In some embodiments, hybrid DNA:RNA origami is scaffolded. In some embodiments, the core structure comprising DNA origami, RNA origami, or hybrid DNA:RNA origami has a specified two-dimensional (2D) or three-dimensional shape.

[0034] As shown in Figure 1, in some embodiments, the core structure 13 is configured to be linked to a capture molecule 2, a detector molecule 1, an 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 with respect to the core nanostructure 13 when linked to it. In some embodiments, any number of one or more core molecules comprises one or more linkers 10, 12, 14 configured to form linkages with the capture molecule 2, the detector molecule 1, and / or the anchor molecule 18. In some embodiments, any number of core molecules is configured to be linked to one or more linkers 10, 12, 14 configured to form linkages with the capture molecule 2, the detector molecule 1, and / or the anchor molecule 18. In some embodiments, one or more linkers are linked to one or more core molecules through chemical bonds. In some embodiments, at least one of the one or more core linkers comprises a core reaction molecule. In some embodiments, each core reaction molecule independently comprises an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-strand nucleic acid of a specific sequence (e.g., RNA or DNA), or 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 trap molecule 2 at a designated first location on the core structure, 2) a detector molecule 1 at a designated second location on the core structure, and optionally 3) an anchor molecule 18 at a designated third location on the core structure. In some embodiments, a designated first core linker 12 is located at the first location on the core structure, and a designated second core linker 10 is located at the 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 specified to maximize intramolecular interactions between the trap 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 captured molecule 2 and the detector molecule 1 that maximizes the intramolecular interaction between the captured molecule 2 and the detector molecule 1.

[0036] As discussed herein, in some embodiments, the distance between the captured molecule 2 and the detector molecule 1 is approximately 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 captured molecule 2 and the detector molecule 1 is approximately 1 nm to approximately 60 nm. In some embodiments, the distance between the captured molecule 2 and the detector molecule 1 is approximately 1 nm to approximately 2 nm, approximately 1 nm to approximately 5 nm, approximately 1 nm to approximately 10 nm, approximately 1 nm to approximately 20 nm, approximately 1 nm to approximately 40 nm, approximately 1 nm to approximately 60 nm, approximately 2 nm to approximately 5 nm, approximately 2 nm to approximately 10 nm, approximately 2 nm to approximately 20 nm, approximately 2 nm to approximately 40 nm, approximately 2 nm to approximately 60 nm, approximately 5 nm to approximately 10 nm, approximately 5 nm to approximately 20 nm, approximately 5 nm to approximately 40 nm, approximately 5 nm to approximately 60 nm, approximately 10 nm to approximately 20 nm, approximately 10 nm to approximately 40 nm, approximately 10 nm to approximately 60 nm, approximately 20 nm to approximately 40 nm, approximately 20 nm to approximately 60 nm, or approximately 40 nm to approximately 60 nm, and includes divisions therein. In some embodiments, the distance between the captured molecule 2 and the detector molecule 1 is approximately 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 40 nm, or 60 nm. In some embodiments, the distance between the captured molecule 2 and the detector molecule 1 is at least approximately 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, or 40 nm. In some embodiments, the distance between the captured molecule 2 and the detector molecule 1 is at most approximately 2 nm, 5 nm, 10 nm, 20 nm, 40 nm, or 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 the first side of the core structure 13, and the optional third location is located on the second side of the core structure 13.

[0038] In some embodiments, capture molecule 2 comprises a protein, peptide, antibody, aptamer (RNA and DNA), fluorescent dye molecule, nanobody, darpin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or a combination thereof. In some embodiments, detector molecule 1 comprises a protein, peptide, antibody, aptamer (RNA and DNA), fluorescent dye molecule, nanobody, darpin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or a combination thereof. In some embodiments, anchor molecule comprises a reaction molecule. In some embodiments, anchor molecule 18 comprises a reaction molecule. In some embodiments, anchor molecule 18 comprises a DNA strand comprising the reaction molecule. In some embodiments, anchor molecule 18 comprises an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single strand nucleic acid of a specific sequence (e.g., RNA or DNA), or polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the anchor molecule 18 comprises a protein, peptide, antibody, aptamer (RNA and DNA), fluorescent dye molecule, nanobody, darpin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or a combination thereof. In some embodiments, a single pair of capture molecule 2 and corresponding detector molecule 1 is linked to the core structure 13. In some embodiments, multiple pairs of capture molecule 2 and corresponding detector molecule 1 are linked to the core structure 13. In some embodiments, the multiple pairs of capture molecule 2 and corresponding detector molecule 1 are spaced apart from each other to minimize crosstalk, i.e., to minimize interaction between capture molecule and / or detector molecule from a first pair and capture molecule and / or detector molecule from a second pair.

[0039] In some embodiments, each component of the supramolecular structure can be independently modified or adjusted. In some embodiments, the step of modifying one or more components of the supramolecular structure can modify the 2D and 3D geometry of the supramolecular structure itself. In some embodiments, the step of modifying one or more components of the supramolecular structure can modify the 2D and 3D geometry of the core structure. In some embodiments, such ability to independently modify components of the supramolecular nanostructure allows for precise control over the organization of one or more supramolecular structures on a solid substrate (e.g., a flat surface) and in a 3D volume (e.g., in a well formed on a solid substrate).

[0040] Capture barcode As shown in Figures 1A and 1B, in some embodiments, the capture molecule 2 is linked to the core structure 13 through the capture barcode 20. In some embodiments, the capture barcode 20 forms a linkage with the capture molecule 2 and also 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 reaction molecule. In some embodiments, the first capture linker 11 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, azide, acridite, single strand nucleic acid of a specific sequence (e.g., RNA or DNA), or 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 reaction molecule. In some embodiments, the second capture linker 6 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, biotin, maleimide, azide, acridite, single-strand nucleic acid of a specific sequence (e.g., RNA or DNA), or 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 terminal, and the second capture linker 6 is attached to the capture bridge 7 at its second terminal. 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 mounting. In some embodiments, the second capture linker 6 is attached to the capture bridge 7 through a physical mounting.

[0041] In some embodiments, the captured barcode 20 is linked to the core structure 13 through a link between a first capture linker 11 and a first core linker 12. In some embodiments described herein, the first core linker 12 is located at a first location on the core structure 13. In some embodiments, the first capture linker 11 and the first core linker 12 are linked to each other through a chemical bond. In some embodiments, the first capture linker 11 and the first core linker 12 are linked to each other by a covalent bond. In some embodiments, the link between the first capture linker 11 and the first core linker 12 is reversible when triggered. In some embodiments, the trigger comprises interaction with a deconstruction molecule ("captured deconstruction molecule", e.g., reference letter 30 in Figures 4 and 7) or exposure to a trigger signal. In some embodiments, the captured deconstruction molecule comprises nucleic acid (DNA or RNA), peptide, small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal includes an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near-infrared illumination.

[0042] In some embodiments, the captured barcode 20 is linked to the captured molecule 2 through a link between the second captured linker 6 and the third captured linker 5, which is bonded to the captured molecule 2. In some embodiments, the third captured linker 5 comprises a reaction molecule. In some embodiments, the third captured linker 5 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-strand nucleic acid of a specific sequence (e.g., RNA or DNA), or polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the third captured linker 5 comprises a DNA sequence domain. In some embodiments, the captured molecule 2 is bonded to the third captured linker 5 through a chemical bond. In some embodiments, the captured molecule 2 is bonded to the third captured linker 5 by a covalent bond. In some embodiments, the second captured linker 6 and the third captured linker 5 are linked to each other through a chemical bond. In some embodiments, the second linker 6 and the third capture linker 5 are linked to each other by a covalent bond. In some embodiments, the link between the second capture linker 6 and the third capture linker 5 is reversible when triggered. In some embodiments, the trigger comprises interaction with a deconstructed molecule ("capture barcode-releasing molecule," e.g., reference letter 31 in Figures 4 and 7) or exposure to a trigger signal. In some embodiments, the capture barcode-releasing molecule comprises nucleic acid (DNA or RNA), peptide, 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, microwave signal, ultraviolet illumination, visible illumination, or near-infrared illumination.

[0043] In some embodiments, upon triggering, 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 a first location. In some embodiments, the capture barcode 20 is configured to provide a signal for detecting the sample molecule when separated from the core structure 13 and the capture molecule 2. In some embodiments, the signal supplied by the capture barcode 20 is a DNA signal.

[0044] Detector barcode As shown in Figure 1A, in some embodiments, the detector molecule 1 is linked to the core structure 13 through the 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 reaction molecule. In some embodiments, the first detector linker 9 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single strand nucleic acid of a specific sequence (e.g., RNA or DNA), or 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 reaction molecule. In some embodiments, the second detector linker 4 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-strand nucleic acid of a specific sequence (e.g., RNA or DNA), or 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 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 terminal, and the second detector linker 4 is attached to the detector bridge 8 at its second terminal. 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 mounting.In some embodiments, the second detector linker 4 is attached to the detector bridge 8 through a physical mounting.

[0045] In some embodiments, the detector barcode 21 is linked to the core structure 13 through a link between a first detector linker 9 and a 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 when triggered. In some embodiments, the trigger comprises interaction with a deconstruction molecule ("detector deconstruction molecule", e.g., reference letter 28 in Figures 4 and 7) or exposure to a trigger signal. In some embodiments, the detector deconstruction molecule comprises nucleic acid (DNA or RNA), peptide, small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal includes 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 the third detector linker 3, which is bonded to the detector molecule 1. In some embodiments, the third detector linker 3 comprises a reaction molecule. In some embodiments, the third detector linker 3 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-strand nucleic acid of a specific sequence (e.g., RNA or DNA), or 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 bonded to the third detector linker 3 through a chemical bond. In some embodiments, the detector molecule 1 is bonded 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 covalent bonds. In some embodiments, the link between the second detector linker 4 and the third detector linker 3 is reversible when triggered. In some embodiments, the trigger comprises interaction with a deconstructed molecule ("detector barcode-releasing molecule," e.g., reference letter 29 in Figures 4 and 7) or exposure to a trigger signal. In some embodiments, the detector barcode-releasing molecule comprises nucleic acid (DNA or RNA), peptide, 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, microwave signal, ultraviolet illumination, visible illumination, or near-infrared illumination.

[0047] In some embodiments, upon triggering, only the link between the first detector linker 9 and the second core linker 10 is broken, thereby breaking the detector molecular linkage with the core structure 13 at the second location. In some embodiments, the detector barcode 21 is configured to provide a signal regarding the detection of the sample molecule when separated from the core structure 13 and the detector molecule 2. In some embodiments, the signal supplied by the detector barcode 21 is a DNA signal.

[0048] Anchor barcode As shown in Figure 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 reaction molecule. In some embodiments, the first anchor linker 15 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, a single strand 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 reaction molecule. In some embodiments, the second anchor linker 17 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, a single strand 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 crosslink 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 terminal, and the second anchor linker 17 is attached to the anchor bridge 16 at its second terminal. 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 a first anchor linker 15 and a 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 when triggered. In some embodiments, the trigger comprises interaction with a deconstruction molecule ("anchor deconstruction molecule," e.g., reference letter 32 in Figures 4 and 7) or exposure to a trigger signal. In some embodiments, the anchor deconstruction molecule comprises nucleic acid (DNA or RNA), peptide, small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal includes 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 reaction molecule, a reaction molecule, a DNA sequence domain, a DNA sequence domain comprising a reaction molecule, or a combination thereof. In some embodiments, the anchor molecule 18 is linked to the second anchor linker 17 through a chemical bond. In some embodiments, the anchor molecule 18 is linked 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 when triggered. In some embodiments, the trigger comprises interaction with a deconstruction molecule ("anchor barcode releasing molecule," e.g., reference letter 33 in Figures 4 and 7) or exposure to a trigger signal. In some embodiments, the anchor barcode releasing molecule comprises 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 receiving 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 molecule, the capture-barcode-release molecule, the detector-deconstruction molecule, and the detector-barcode-release molecule are of the same type. In some embodiments, the capture-deconstruction molecule, the capture-barcode-release molecule, the detector-deconstruction molecule, and the detector-barcode-release molecule are of different types. In some embodiments, the capture-deconstruction molecule, the capture-barcode-release molecule, the detector-deconstruction molecule, the detector-barcode-release molecule, the anchor-deconstruction molecule, and the anchor-barcode-release molecule are of the same type. In some embodiments, the capture-deconstruction molecule, the capture-barcode-release molecule, the detector-deconstruction molecule, the detector-barcode-release molecule, the anchor-deconstruction molecule, and the anchor-barcode-release molecule are of different types. In some embodiments, any combination of the capture-deconstruction molecule, the capture-barcode-release molecule, the detector-deconstruction molecule, the detector-barcode-release molecule, the anchor-deconstruction molecule, and the anchor-barcode-release molecule is of the same type.

[0053] 3-arm nucleic acid junction-based supramolecular structure Figures 2 and 3 provide illustrative diagrams of a supramolecular structure 40 having a three-arm nucleic acid junction and associated dependent components. Figure 2 provides the complete supramolecular structure, while Figure 3 provides the dependent components comprising the supramolecular structure of Figure 2. In some embodiments, the dependent components of the supramolecular structure comprise five DNA strands (reference letters 20-24), one DNA strand 25 having terminal modification regions, and two antibodies (1, 2) modified by single DNA linkers 3, 5. Figure 4 provides illustrative diagrams of each deconstruction molecule configured to cleave each dependent component from the supramolecular structure 40 of Figure 2. Reference letters 1-18 in Figures 2-4 correspond to the respective components provided in Figure 1A with the same reference letters.

[0054] TIFF0007899273000001.tif22170

[0055] In some embodiments, the first core strand 23 of the core structure comprises a first core linker 12 having a DNA sequence domain. In some embodiments, the first core strand 23 is labeled "A" in Figures 2-4 and 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 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 having 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 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 towhold ("TH"). In some embodiments, the capture barcode sequence 7 comprises a towhold ("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. 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] TIFF0007899273000002.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 having 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 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 towhold ("TH"). In some embodiments, the detector barcode sequence 8 comprises a towhold ("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. 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, a third core linker 14 is complementary to a first anchor linker 15 on an anchor barcode strand 22. In some embodiments, the anchor barcode strand 22 comprises a DNA strand having 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 singular anchor barcode sequence 16 between the first anchor linker 15 and the second anchor linker 17. In some embodiments, the singular anchor barcode sequence 16 comprises nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the singular anchor barcode sequence 16 comprises a polymer such as PEG. In some embodiments, the anchor barcode 22 comprises a short domain called a towhold ("TH"). In some embodiments, the anchor barcode sequence 16 comprises a towhold ("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 to a terminal modification portion 34 25. In some embodiments, the terminal modification portion 34 comprises a reaction molecule. In some embodiments, the terminal modification portion 34 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-strand nucleic acid of a specific sequence (e.g., RNA or DNA), or polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators).

[0063] Figure 4 provides exemplary embodiments of deconstruction molecules that can be used to trigger different reactions to the supramolecular structure 40. In some embodiments, the detector deconstruction molecule 28 comprises a TH domain on the detector barcode 21 and a TH' domain having a complementary arrangement to 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 break the link between the detector barcode 21 and the core structure (e.g., the second core strand 24). In some embodiments, the detector barcode release molecule 29 comprises a TH domain on the detector barcode 21 and a TH' domain having a complementary arrangement to the third detector linker 3 (e.g., a DNA sequence domain). In some embodiments, the detector barcode release molecule 28 is configured to break 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 break the link between the capture barcode 20 and the core structure (e.g., the first core strand 23). In some embodiments, the capture-barcode-release 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-release molecule 31 is configured to break 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 on the anchor barcode 22 and a TH' domain having a complementary arrangement to 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 break the link between the anchor barcode 22 and the core structure (e.g., the second core strand 24). In some embodiments, the anchor barcode release molecule 33 comprises a "TH" domain on the anchor barcode 22 and a "TH'" domain having a complementary arrangement to the anchor molecule 18 (e.g., a DNA sequence domain). In some embodiments, the anchor barcode release molecule 33 is configured to break the link between the anchor barcode 22 and the anchor molecule 18.

[0066] TIFF0007899273000003.tif22170

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

[0068] In some embodiments, the core structure 13 comprises scaffold DNA origami, in which circular ssDNA molecules called "scaffold" strands fold into a predetermined 2D or 3D shape by interacting with two or more shorter ssDNAs called "staple" strands, which interact with specific subsections of the ssDNA "scaffold" strands.

[0069] As shown in Figures 5 and 6, in some embodiments of the supramolecular structure, the core structure 13 comprises DNA origami. In some embodiments, the core structure 13 comprises a first core linker 12 comprising a DNA sequence domain. In some embodiments, the first core linker 12 is complementary to a first capture linker 11 on a capture barcode strand 20. In some embodiments, the capture barcode strand 20 comprises a DNA strand having either the first capture linker 11 or a 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 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. 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 having 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 having 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 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 towhold ("TH"). In some embodiments, the unique detector barcode array 8 includes 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. 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 having a DNA sequence domain. In some embodiments, the third core linker 14 is complementary to a first anchor linker 15 on the anchor barcode strand 22. In some embodiments, the anchor barcode strand 22 comprises a DNA strand having 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 singular anchor barcode sequence 16 between the first anchor linker 15 and the second anchor linker 17. In some embodiments, the singular detector barcode sequence 16 comprises nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the singular detector barcode sequence 16 comprises a polymer such as PEG. In some embodiments, the anchor barcode 22 comprises a short domain called a towhold ("TH"). In some embodiments, the anchor barcode array 16 includes 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 to a terminal modification portion 34. In some embodiments, the terminal modification portion 34 comprises a reaction molecule. In some embodiments, the terminal modification portion 34 comprises a reaction molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-strand nucleic acid of a specific sequence (e.g., RNA or DNA), or 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 associated core linker 14, anchor linker 15, barcode 16, or anchor linker 17, and the core structure 13 directly interacts with or comes into contact with the substrate discussed herein.

[0075] Figure 6 provides exemplary embodiments of deconstruction molecules that can be used to trigger different reactions to the supramolecular structure 40. In some embodiments, the detector deconstruction molecule 28 comprises a TH domain on the detector barcode 21 and a TH' domain having a complementary arrangement to a 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 break the link between the detector barcode 21 and the core structure 13. In some embodiments, the detector barcode release molecule 29 comprises a TH domain on the detector barcode 21 and a TH' domain having a complementary arrangement to a third detector linker 3 (e.g., a DNA sequence domain). In some embodiments, the detector barcode release molecule 28 is configured to break 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 on the capture barcode 20 and a TH' domain having a complementary arrangement to the 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 break the link between the capture barcode 20 and the core structure 13. In some embodiments, the capture-barcode-release molecule 31 comprises a TH domain on the capture barcode 20 and a TH' domain having a complementary arrangement to the third capture linker 5 (e.g., a DNA sequence domain). In some embodiments, the capture-barcode-release molecule 31 is configured to break 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 on the anchor barcode 22 and a TH' domain having a complementary arrangement to the 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 break the link between the anchor barcode 22 and the core structure 13. In some embodiments, the anchor barcode release molecule 33 comprises a TH domain on the anchor barcode 22 and a TH' domain having a complementary arrangement to the anchor molecule 18 (e.g., a DNA sequence domain). In some embodiments, the anchor barcode release molecule 33 is configured to break 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 predetermined based on the function of individual supramolecular structures that remain structurally intact when exposed to 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 different components that are part of the supramolecular structure remain physically linked to one another even after exposure to a deconstruction molecule and / or a trigger signal. In some embodiments, when the supramolecular structure is in an unstable state, predetermined sections of the supramolecular structure (e.g., one or more dependent components) are physically cut, i.e., released (separated) from the supramolecular structure as a result of exposure to a deconstruction molecule and / or a trigger signal. In some embodiments, the supramolecular structure is configured to shift from a stable state to an unstable state upon interaction with a sample molecule (as described herein). In some embodiments, the supramolecular structure is configured to shift from an unstable state to a stable state upon interaction with a sample molecule (as described herein). In some embodiments, the sample molecule that triggers the state change of the supramolecular structure comprises a protein, a protein cluster, a peptide fragment, a peptide fragment cluster, 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 an unstable state configuration includes a physical state that can sever the link between the core nanostructure 13 and the captured molecule 2 so that the captured molecule 2 is released from the core nanostructure 13. In some embodiments, the unstable state configuration includes a physical state that can sever the link between the core nanostructure 13 and the detector molecule 1 so that the detector molecule 1 is released from the core nanostructure 13. In some embodiments, the unstable state configuration includes a physical state that can sever the link between the core nanostructure 13 and the captured molecule 2 and the link between the core nanostructure 13 and the detector molecule 1 so that the captured 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 captured molecule 2, 2) the detector molecule 1, or 3) both of these is severed when a trigger (e.g., a deconstruction molecule or a trigger signal as described herein) is received. Figure 8 provides an exemplary diagram of a supramolecular structure 40 in an unstable state, initially with the detector molecule 1 coupled to the core structure 13 through a linkage with the detector barcode 21. Continuing with the reference to Figure 8, the interaction with the deconstruction molecule 42 (e.g., detector deconstruction molecule 28) then breaks the link between the detector barcode 21 and the core structure 13, thereby freeing the detector molecule 1 from the core nanostructure 13. In some embodiments, in the unstable state, the trapped molecule 2 on the core nanostructure 13 and the detector molecule 1 diffuse freely to each other, unconstrained except 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 when the link between the core structure 13 and the capture molecule 2 is broken. In some embodiments, the stable state configuration comprises a physical state in which the detector molecule 1 remains bound to the core nanostructure 13 when the link between the core structure 13 and the detector molecule 1 is broken. In some embodiments, the stable state configuration comprises a physical state in which the capture molecule 2 and the detector molecule 1 are positioned close to each other. In some embodiments, the detector molecule 1 and the capture molecule 2 are positioned close to each other with or without the formation of a clear bond between them. 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 them (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 a sample molecule 44 from a sample (described herein). Figure 9 provides an exemplary diagram of a stable supramolecular structure 40 in which the capture molecule 2 is linked to the detector molecule 1 through a linkage with the sample molecule 44. Continuing the reference to Figure 9, 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 the linkage with the capture molecule 2. As will be further described 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 sample molecules from the sample. In some embodiments, interaction with the deconstruction molecule and / or the trigger signal does not break the link between the capture molecule and the detector molecule.

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

[0082] Figure 11 provides an exemplary embodiment of a supramolecular structure 40 that shifts from a stable state to an unstable state. As described herein, when the supramolecular structure 40 is in a stable configuration, the detector molecule 1 remains linked to the core structure 13 due to the fact that the detector molecule 1 is linked to the capture molecule 2 when interacting with the corresponding deconstruction molecule and / or trigger signal. Continuing the reference to Figure 11, in some embodiments, interaction with a sample molecule 44 from the sample breaks the link between the capture molecule 2 and the detector molecule 1, causing the supramolecular structure to transition to an unstable state in which the detector molecule 1 is linked only to the core nanostructure 13 through linkage with the detector barcode 21. In some embodiments, the sample molecule 44 comprises a single molecule. In some embodiments, instead, the sample molecule comprises multiple sample molecules. In some embodiments, instead, the sample molecule comprises a molecular cluster. In some embodiments, as described herein and shown in Figure 11, when the supramolecular structure is in an unstable state, the interaction with the corresponding deconstruction molecule 42 breaks the link between the core structure 13 and the detector barcode 21, thereby freeing (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 a sample molecule 44 that binds to the detector molecule 1, thereby breaking the link between the capture molecule 2 and the detector molecule 1. The capture molecule 2 is then released from the core structure 13 upon interaction with the corresponding deconstruction molecule 42 (e.g., the capture-deconstruction molecule 30).

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

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

[0086] In some embodiments, multiple sample molecules in a sample are detected simultaneously by multiplexing, in which case the multiple supramolecular structures supply multiple signals (e.g., detector barcodes, capture barcodes) for sequence analysis and sample identification. In some embodiments, the method for detecting samples in a sample described herein provides high throughput and high multiplexing capabilities by using multiple supramolecular structures. In some embodiments, the high throughput and high multiplexing capabilities provide high precision for the detection and quantification of sample molecules. In some embodiments, the method for detecting samples in a sample described herein is 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 errors due to cross-reactivity relationships. In some embodiments, such errors due to cross-reactivity relationships involve the interaction (e.g., intermolecular interactions) between capture molecules and / or detector molecules of one supramolecular structure and capture molecules and / or detector molecules of another supramolecular structure. In some embodiments, the core structures of the multiple supramolecular structures are identical to each other. In some embodiments, the structural, chemical, and physical properties of each supramolecular structure are clearly designed. In some embodiments, the same core structure has a specified shape, size, molecular weight, 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, that limits cross-reactivity between supramolecular structures. In some embodiments, all core structures have the same molecular weight and are precise down to the purity of the core molecules. In some embodiments, each core structure has at least one capture molecule and at least one corresponding detector molecule.

[0087] In some embodiments, since the state change (from unstable to stable) is primarily driven through intramolecular interactions (between the capture molecule and the detector molecule on the same supramolecular structure), multiple supramolecular structures interact independently with different sample molecules from the sample. In some embodiments, multiple supramolecular structures may share structural similarities due to having the same certain dependent components, but the interaction between the sample molecule from the sample and the supramolecular structure is predetermined by the corresponding capture molecule and detector molecule. In some embodiments, each pair of detector molecule and capture molecule on a given supramolecular structure interacts specifically with a particular sample molecule in the sample, and a state change of the supramolecular structure can be achieved upon interaction with such a specific sample molecule. In some embodiments, each supramolecular structure is provided with a unique DNA barcode corresponding to each pair of detector molecule and capture molecule. In some embodiments, each pair of detector molecule and capture molecule on a given supramolecular structure is designed to interact with more than one sample molecule in the 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 within a typical composite biological sample. In some embodiments, single-molecule sensitivity comprises a capture molecule and a detector molecule of a given supramolecular structure configured to shift from an unstable state to a stable state (or vice versa) upon binding with a single sample molecule. In some embodiments, multiple supramolecular structures limit or eliminate non-specific interactions and the sample manipulation required to reduce any user-induced errors.

[0089] In some embodiments, multiple supramolecular structures are mounted on one or more solid substrates, e.g., planar or patterned substrates. Figure 12 provides an exemplary method for detecting one or more sample molecules in a sample using one or more supramolecular structures. In some embodiments, a sample comprising one or more samples (e.g., sample pool 102) is brought into contact with one or more supramolecular structures 40 (e.g., supramolecular structure pool 100). In some embodiments described herein, multiple supramolecular structures are given mounted on one or more solid substrates for single-molecule organization provided herein. In some embodiments, the sample is in contact with the supramolecular structures for a period of time from about 30 seconds to about 24 hours. In some embodiments, the sample is left at a constant temperature with the supramolecular structures for a period of time from about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, and about 24 hours to about 48 hours.

[0090] Continuing with the reference to Figure 12, in some embodiments, all of these supramolecular structures are in an unstable state (as shown by reference letter 100). In some embodiments described herein, the interaction between the sample molecule and the corresponding capture molecule 2 and detector molecule 1 shifts each supramolecular structure from an unstable state to a stable state (e.g., a sandwich configuration of capture molecule, sample molecule, and detector molecule shown by reference letter 104). In some embodiments, certain types of sample molecules will bind to specific pairs of capture molecule and detector molecule. In some embodiments, a given pair of capture molecule and detector molecule is configured to bind to more than one type of sample molecule. In some embodiments, the switching from an unstable state to a stable state for any given supramolecular structure depends on the specific capture molecule and detector molecule bound to that supramolecular structure and the sample molecule in the sample. In some embodiments, considering that the state changes of the supramolecular structure depend primarily on the intramolecular structure (the components located on the supramolecular structure), potential intermolecular interactions between two different supramolecular structures are minimized or eliminated by limiting the net concentration of the supramolecular structure 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 trapping molecules and detector molecules on a given supramolecular structure.

[0091] As can be seen in reference letter 104 in Figure 12, after contact 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 molecule, the sample molecule, and the detector molecule are simultaneously linked to each other) through interaction with the sample molecule, while at least one of the supramolecular structures remains in an unstable state because the respective capture molecule and detector molecule are not bound to or interacting with the sample molecule from the sample.

[0092] After the sample has been in contact with the supramolecular structure for a specified amount of time, the mixed solution of the sample and the supramolecular structure shown in Figure 12 is triggered to break the link between the detector molecule and the core structure (reference letter 106). In some embodiments, the trigger includes the step of introducing a solution comprising one or more deconstructed molecules (e.g., the detector deconstructed molecule reference letter 28 in Figures 4 and 7) into the mixed solution. In some embodiments, the trigger includes the step of exposing the mixed solution to a trigger signal. In some embodiments, the trigger includes a combination of the steps of introducing the deconstructed molecules into the mixed solution and exposing the mixed solution to a trigger signal. In some embodiments described herein, the deconstructed molecules include nucleic acids (DNA or RNA), peptides, small organic molecules, or combinations thereof. In some embodiments described herein, the trigger signal includes 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 left at a constant temperature for a specified amount of time with one or more deconstructed molecules. In some embodiments, the mixed solution is left at a constant temperature with the deconstructing molecules for a period of about 30 seconds to about 24 hours. In some embodiments, the mixed solution is left at a constant temperature with the deconstructing molecules for a period of about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, and about 24 hours to about 48 hours.

[0093] As shown by reference numeral 106 in Figure 12, in some embodiments, the step of triggering the mixed solution severs the link between the detector molecules and the core structure of the supramolecular structure, for example, the link between the detector barcode (e.g., reference numeral 21 in Figure 1) and the core structure 13. In some embodiments, the severance is achieved by substitution of nucleic acid (DNA / RNA) strands, optical severance, chemical severance, another technique known in the art, or a combination thereof. With respect to the supramolecular structure shifted to a stable state, the detector molecules 1 remain linked to the core structure 13 through linkage with the corresponding capture molecule 2. With respect to the supramolecular structure remaining in an unstable state, the detector molecules 112 released from each supramolecular structure are shown. In some embodiments, the released detector molecules 1 remain linked to their respective detector barcodes 21.

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

[0095] In some embodiments, after the release detector molecules are separated from the mixed solution, the detector barcode 21 is cleaved from the corresponding detector molecule linked to each capture molecule (e.g., positioned on a supramolecular structure shifted to a stable state). In some embodiments, the detector barcode 21 is cleaved from the corresponding detector molecule by substitution of nucleic acid (DNA / RNA) strands, optical cleavage, chemical cleavage, or a combination thereof. In some embodiments, the detector barcode is 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 release molecule (e.g., reference letter 29 in Figures 4 and 7).

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

[0097] In some embodiments, the cleaved detector barcodes provide signals correlated to each sample molecule bound to each detector molecule. In some embodiments described herein, the detector barcodes comprise DNA strands. In some embodiments, the detector barcodes provide DNA signals correlated to sample molecules. In some embodiments, as shown by reference numeral 110 in Figure 12, the isolated detector barcodes 21 are analyzed to identify and / or quantify the corresponding sample molecules in the sample. In some embodiments, the analysis of the isolated detector barcodes comprises genotyping, qPCR, sequencing, or a combination thereof.

[0098] In some embodiments, the method for detecting sample molecules shown in Figure 12 comprises the step of cleaving the capture barcode 20 from the corresponding capture molecule linked to each detector molecule (e.g., positioned on a supramolecular structure shifted to a stable state). In some embodiments, the capture barcode 20 is cleaved from the corresponding detector molecule by substitution of nucleic acid (DNA / RNA) strands, optical cleavage, chemical cleavage, or a combination thereof. In some embodiments, the detector barcode is 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 letter 31 in Figures 4 and 7).

[0099] In some embodiments, the cleaved captured barcode 20 is isolated from the solution comprising the supramolecular structure (reference letter 108 in Figure 12). In some embodiments, the cleaved captured barcode 20 is isolated from the solution by precipitation of polyethylene glycol (PEG). In some embodiments, the cleaved captured barcode 20 is isolated from the solution by binding the core structure in the solution to a solid support through corresponding anchor molecules on each core structure, followed by isolation of the cleaved captured barcode by centrifugation, microfiltration, chromatography, or a combination thereof.

[0100] In some embodiments, the cleaved capture barcodes provide signals correlated to each sample molecule bound to each detector molecule. In some embodiments described herein, the capture barcodes comprise DNA strands. In some embodiments, the capture barcodes provide DNA signals correlated to sample molecules. In some embodiments, as shown by reference numeral 110 in Figure 12, the isolated capture barcodes 21 are analyzed to identify and / or quantify the corresponding sample molecules in the sample. In some embodiments, the analysis of the isolated capture barcodes comprises genotyping, qPCR, sequencing, or a combination thereof.

[0101] Detection of sample molecules using surface assays Figure 13 provides an illustrative diagram of a technique for detecting sample molecules in a sample using a surface-based assay that utilizes a supramolecular structure described herein for single-molecule counting of sample molecules in the sample. In some embodiments, the supramolecular structure comprises a DNA origami core. In some embodiments, a planar substrate 400 is provided, comprising (a) a reference marker 402 that acts as a reference coordinate for all feature areas on the substrate 400, (b) a predetermined set of micropatterning binding sites 406 that can immobilize individual core structures (e.g., DNA origami), and (c) a background passivation area 404 that minimizes or prevents interaction between the surface of the substrate 400 and the supramolecular structure (comprising capture molecules 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 include substrates having micropatterning wells or protrusions on the surface. In some embodiments, the reference marker comprises a geometric feature area predetermined on the surface and used as a reference feature area for other feature areas on the substrate. In some embodiments, the reference marker 402 is coated with a core structure of a supramolecular structure (e.g., DNA origami) or a polymer or self-assembled monolayer that does not interact with other molecules. In some embodiments, the background passivation area 404 minimizes or prevents interaction between the surface of the substrate 400 and the sample molecules of the sample. In some embodiments, the planar substrate 400 includes optical or electrical devices such as FETs, ring resonators, photon crystals, or microelectrodes that are predetermined before 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 bonding sites 406 is at least about 40 nm to about 5000 nm.In some embodiments, the shape of the binding site 406 is circular, square, triangular, or other polygonal. In some embodiments, the chemical group used for the passivation site 404 is a neutrally charged molecule such as trimethylsilyl (TMS), an uncharged polymer such as PEG, a zwitterionic polymer, or a combination thereof. In some embodiments, the chemical group used to define the binding site 406 is a silanol group, a carboxyl group, a thiol group, another group, or a combination thereof.

[0102] In some embodiments, a single supramolecular structure 40 is attached to each binding site 406 (Step 1). Reference numeral 416 provides diagrams of the components of the supramolecular structure 40 individually and assembled and arranged on a planar substrate (the components are described herein, for example, in Figures 1, 2-3, and 5-6). In some embodiments, the supramolecular structure 40 comprises a core structure 13 comprising DNA origami, which is attached to each of the binding sites using DNA origami placement techniques (Step 1). In some embodiments, the supramolecular structure 40 is assembled before being attached to each binding site 406. In some embodiments, the DNA origami has a unique shape and dimensions that facilitate binding to the binding sites using DNA origami placement techniques. In some embodiments, the DNA origami placement comprises a directed self-assembly technique for organizing individual DNA origami (e.g., core structures) on a surface (e.g., a micro-patterned surface). In some embodiments, instead of DNA origami arrangement, the reactive groups of the supramolecular nanostructure 40 are bound to pre-organized DNA origami on the binding site. In some embodiments, both of these methods for binding the supramolecular nanostructure to the corresponding binding site rely on the ability to organize one or more molecules on the binding site of micropatterning using DNA origami arrangement techniques. In some embodiments, the planar substrate is considered to be able to be stored in a clean environment for a considerable period of time after this step.

[0103] Continuing with the reference to Figure 13, in some embodiments, a sample comprising the sample molecule (as described herein) is brought into contact with a planar substrate (Step 2). In some embodiments, the sample is brought into contact with the planar substrate using a flow cell. In some embodiments, the sample is kept at a constant temperature on the planar substrate to which the supramolecular structure is attached at the binding site 406. In some embodiments, the keeping at a constant temperature period can range from about 30 seconds to about 24 hours. In some embodiments, the keeping at a constant temperature period can range from about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, and about 24 hours to about 48 hours.

[0104] In some embodiments, sample molecules 44 in the sample interact with a supramolecular structure 40 on a flat surface 400. In some embodiments, a single copy of a particular sample molecule 44 binds simultaneously to both the capture molecule and the detector molecule so that the particular supramolecular structure switches from an unstable state to a stable state 418 (as described herein, for example, in Figures 8-10). In some embodiments, a single copy of a particular sample may simultaneously interact with the capture molecule and the detector molecule, which are already bound to each other, to switch the supramolecular structure from a stable state to an unstable state (as described herein, for example, in Figure 11).

[0105] Continuing with the reference to Figure 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 in Figure 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 nucleic acid (DNA or RNA), peptide, small organic molecule, or a combination thereof. In some embodiments described herein, the trigger signal comprises an electrical signal, microwave signal, ultraviolet illumination, visible illumination, or near-infrared illumination. In some embodiments, the deconstruction molecule associated with a supramolecular structure mounted on the planar substrate is enabled 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 left at a constant temperature with the supramolecular structure for about 30 seconds to about 24 hours (step 3). In some embodiments, the constant temperature storage period can be approximately 30 seconds to approximately 1 minute, approximately 1 minute to approximately 5 minutes, approximately 5 minutes to approximately 30 minutes, approximately 30 minutes to approximately 1 hour, approximately 1 hour to approximately 5 hours, approximately 5 hours to approximately 12 hours, approximately 12 hours to approximately 24 hours, or approximately 24 hours to approximately 48 hours.

[0106] In some embodiments, interaction with deconstructed molecules cleaves the detector molecules and detector barcodes of all unstable supramolecular structures, thereby physically severing these detector molecules and detector barcodes from the planar substrate 400. In some embodiments, the physically severed detector molecules and detector barcodes are removed during one or more buffer washes at the end of the constant temperature setting stage. In some embodiments, where the supramolecular structure on the planar substrate has shifted to a stable state due to the capture of a single sample molecule, the corresponding detector molecules and detector barcodes remain linked to the supramolecular structure 420, which are stably bound to the planar substrate due to a sample-intervened sandwich formed between the corresponding detector molecule and the capture molecule (i.e., a link between the capture molecule, the sample molecule, and the detector molecule).

[0107] Continuing with the reference to Figure 13, in some embodiments, the detector barcode located at the site of the supramolecular structure shifted to a stable state is used as a binding site 422 to the signaling element 414 (Step 4). In some embodiments, the signaling element comprises a fluorescent molecule or microbeads, 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 a planar substrate. In some embodiments, the signaling elements are 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 a hybridization chain reaction.

[0108] In some embodiments, the introduction of the signaling element 414 described in step 4 results in the presence of the signaling element at each sample location as a result of all individual sample capture events (i.e., linkages between the capture molecule, the detector molecule, and the sample molecule) (linked with the capture molecule and the detector molecule). In some embodiments, the signaling element is optically active and can be measured using a microscope or a built-in optical sensor in the planar substrate 400. In some embodiments, the signaling element is electrically active and can be measured using a built-in electrical sensor. In some embodiments, the signaling element is magnetically active and can be measured using a built-in magnetic sensor. In some embodiments, each signal event is determined by the corresponding detector molecule and capture molecule with respect to the capture of the same type of sample molecule (a single copy of the same type of sample molecule), and therefore, the step of counting the number of locations where the signaling element is present gives quantification of the sample molecule in the sample.

[0109] In some embodiments, the method for detecting a sample, as described in Figure 13, uses a supramolecular core in which the core structure is bonded to DNA origami that has already been organized on the surface of a planar substrate through the anchor portion of the core structure.

[0110] In some embodiments, the method for detecting a sample, as described in Figure 13, enables the detection of a single type of sample molecule. In some embodiments, the method for detecting a sample, as described in Figure 13, enables the detection of multiple types of sample molecules (multiplex sample molecule detection). In some embodiments, each supramolecular structure is barcoded to specifically identify its respective capture molecule and detector molecule, thereby enabling the identification of each capture sample molecule. In some embodiments, each supramolecular structure is barcoded using its respective anchor molecule.

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

[0112] In step 530, a passivation layer 532, for example, a passivation polymer, is added, and the passivation layer 532 reacts only with the upper layer reactive groups 522. The passivation polymer can be entropic, for example, made from groups that specifically react with the upper layer reactive groups 522 and not with the binding layer reactive groups 524. The upper surface of the upper layer 506 comprises the passivation layer 532 and surrounds the binding site 542. In step 540, a supramolecular structure 40, such as DNA origami 540, is added to the binding site 542, and interacts with the reactive groups 524 of the binding site 542 so that each binding site 542 of the substrate 550 comprises a supramolecular structure 40. In embodiments, it should be understood that the substrate 550 can be considered to have supramolecular structures 400 within each binding site 542 if, within a certain tolerance range, for example, more than 95% or more than 97% of the binding sites 542 comprise at least one supramolecular structure 40. Furthermore, certain binding sites 542 can be reserved for reference or control purposes. In some embodiments, each binding site 542 comprises up to one or a single supramolecular structure 40. The binding sites 542 may have a predetermined shape and size achieved by patterning techniques of the upper layer 506. The supramolecular structures 40 can be arranged as pre-formed units or assembled stepwise on the binding sites 542. In one example, a core structure, such as a DNA origami moiety, can be initially associated with the binding site 542. Following the association, the capture molecule and detector molecule can be linked to their respective locations at desired predetermined intervals in an unstable state.

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

[0114] Figure 15 shows an example of technique 600 for forming or manufacturing a substrate such as the substrate 400 shown in Figure 13. Technique 600 is initiated using an initiating base layer 602 and a bonding layer 604. The base layer 602 is a substantially flat layer on 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 the surface of the base layer 602. The illustrated example has the bonding layer 604 on only one side of the base layer 602, but in addition to or instead of this, the bonding layer 604 may be present on the opposite surface of the base layer 602 to increase the reaction area of ​​the substrate.

[0115] Next, a thin film 610 of the material (metal, metal oxide, polymer, or any material desirable for patterning) is deposited on the bonding layer 604. In embodiments, an adhesion promoter may be added to the upper layer to improve the bonding of the thin film 610. In embodiments, the thickness of the thin film 610 can be in the range of 2 angstroms to 10 microns. The thickness of the thin film 610 will affect the topography of the final patterned substrate. The thin film 610 can be patterned using conventional methods such as photolithography, nanoimprint lithography, direct writing, or roll-to-roll embossing (generally, lithography may include a pattern transfer step, not shown, which may involve etching the film). After etching of the thin film 610, the lower surface 620 of the bonding layer 604 is exposed. Having a laminate of thin films 610, it is possible to etch through specific layers to expose various materials (chemical reactions). Additional procedures such as activation may be performed on the lower surface 620 to generate bonding sites 640. Next, the supramolecular structure 40, such as DNA origami, is arranged on the patterned surface.

[0116] The dimensions of the origami are designed deductively from the arrangement that determines how many supramolecular structures 40 are placed in the capture site. The dimensions of each binding site 640 across the surface 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 boundary of the xy dimensions is predetermined by the wall 650. Thus, the physical space of the well can act as a barrier to more than one supramolecular structure 40 associated with the surface 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 dimensions 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 dimensions of the binding site across the xy plane. If the binding site 640 is a circle, the supramolecular structure 40 or the core structure may have at least one dimension that is larger than the radius of this 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 the patterning takes place. Exemplary modifications may include the growth of SAMP (self-assembled monolayer phosphonate) or silane treatment deposited by conventional methods such as chemical vapor deposition. The surface chemistry (functional groups on the surface of the patterned wafer) differs between the thin film 610 (or modified thin film 610) and the SiO2 underlayer surface 620 (or any other film that may have been deposited before the thin film 610) exposed after patterning.

[0118] Figure 16 shows an example of a technique 700 for forming or manufacturing a substrate such as the substrate 400 shown in Figure 13, comprising a base layer 702, a binding layer 704, and a thin film 710. The binding sites can be formed as shown in Figure 15. In this case, the thickness 720 of the thin film 710 is sufficiently greater than the thickness of the DNA origami core structure 13 of the supramolecular structure 40. For example, the thin film 610 is 100 nm thick, and the origami of the core structure 13 is 1 nm thick. The core structure is placed in the wells of the binding sites 640 in a one-to-one correspondence. This arrangement can be achieved by using link groups between the origami and complements specifically deposited or activated on the upper surface 750 of the binding sites 740 at the bottom of the wells. Following loading an excess amount of origami into this array, origami linking can be performed, after which washing is performed to remove all but one of the origami from each well.

[0119] The thin film 710 can be used for three-dimensional control of the cargo element 770. The features of the joint can include x and y dimensions extending across the top surface 750 and dimensions corresponding to the thickness 720 of the thin film 710.

[0120] Figure 17 shows an example of technique 800 for forming or manufacturing a substrate such as the substrate 400 shown in Figure 13. Technique 800 is initiated using an initiating base layer 802 and a bonding layer 804. The base layer 802 is a substantially flat layer on 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 wafer or a silicon wafer, and the bonding layer 804 is silicon dioxide, silicon nitride, graphene, or silicon carbide grown on the 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 absent. The illustrated example has the bonding layer 804 on only one side of the base layer 802, but in addition to or instead of this, the bonding layer 804 may 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. This sacrificial film 810 can be a photoresist, nanoimprint resist, metal, or a similar material that has the function of being patterned. The sacrificial film 810 is patterned to provide herein a desired pattern corresponding to the desired bonding site pattern. That is, the sacrificial film 810 that remains after patterning corresponds to the final location of the bonding sites. The sacrificial film 810 is used as an etching mask to transfer this pattern into the bonding layer 804. The pattern transfer process can be carried out using reactive ion etching (plasma etching) or solution phase etching. The etching depth can be controlled by the exposure time of the material to the etching solution or by striking the underlying layer. After that, the sacrificial film 810 will be removed as shown in the figure. Next, a conformal coating 830 is deposited on top of the bonding layer 804, and the conformal coating 830 fills the etched or formed features achieved in the preceding step. This deposition can be carried out using sputtering, atomic layer deposition, electron beam deposition, etc. The thickness of this new conformal coating 830 is greater than the depth of the features etched in the preceding stage so that these features are not exposed in subsequent stages. The surface is planarized, for example, by CMP (chemical mechanical polishing) or other means to expose the predetermined topography in previous stages. Next, the supramolecular structures 40 are arranged on the patterned surface. The dimensions of the supramolecular structures 40 are designed deductively from the arrangement which determines how many origamis are placed at the bonding sites 840. The chemical action of the conformal coating 830 can be modified by the growth of a passivation film nucleated from the surface before the arrangement is performed. Exemplary modifications can be by the growth of SAMP (self-assembled monolayer phosphonate) or by silane treatment deposited by conventional methods such as chemical vapor deposition. The planar chemical interactions (functional groups on the surface of the patterned wafer) differ between the conformal coating film 830 (or modified conformal coating film 830) and the bonding layer 804 (or other films that may have been deposited before the conformal coating film 830) that are exposed after patterning.

[0122] Figure 18 illustrates an example of a supramolecular assembly process for sample detection. In some embodiments, a core structure 13 (e.g., DNA origami) is placed on binding sites 910 on a patterned substrate 920 before being assembled as a supramolecular structure 40. Thus, each core structure 13, once placed on its individual binding sites 910, can be specialized to possess desired sample detection characteristics 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 such links can be specialized to be in an unstable configuration in the absence of a sample. That is, the step of linking to the capture / detection molecule can be performed after the placement of the core structure 13. Furthermore, various linkers having specific triggers and specific lengths can be selected depending on the desired three-dimensional properties. As described above, these linkers can be individualized for each binding site so that each binding site 910 on the patterned substrate 920 has known sample detection characteristics that can be the same as 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. Furthermore, each core structure 13 can be bound to a barcode sequence, or each core structure 13 can comprise a barcode sequence.

[0124] Furthermore, the detection system may be configured to detect, in addition to or instead of, electrical, optical, and / or magnetic environmental changes unique to the sample bound to the supramolecular structure 40. Such detection can be worked in conjunction with amplification-based detection to generate information indicating the presence and / or level of the sample, as well as the identity of the captured molecule and / or detector molecule. Moreover, capture and detector linking can be patterned or associated with known locations or binding sites.

[0125] The detection system 950 may comprise an input / output circuit 952, a display 954, and a processor-based controller 956 that executes instructions stored in memory. Detection can be configured via the display 954 to generate notifications or information regarding sample binding relating to the migration of a 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 sample 44 can be detected by a field-effect transistor device as shown in Figure 19. Thus, the detection system 950 can have the ability to detect environmental changes unique to the sample binding 44, which are indicated as changes in detection electrical signals 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 by multiple binding sites 1004 coupled to a single electrical lead 1002. The detection signals are supplied 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 sample 44 can be detected by a metal oxide semiconductor image sensor device shown in Figure 20. Thus, the detection system 950 can have the ability to detect environmental changes unique to sample binding 44, 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. An optical conductor 1130 extends through the passivation layer 1102 and the metal oxide layer 1100 and is coupled to a binding site 1140, enabling the detection of optical changes unique to sample binding by a photodetector 1150. Each binding site 1120 can be coupled to a dedicated optical conductor 1130 and a photodetector 1150. The detection signal is supplied to the detection device 950 for analysis. It should be understood that the detection methods disclosed in this invention are illustrative and other embodiments are anticipated.

[0128] Circuit board recycling A key feature of the techniques disclosed in this invention is that the surface of an organized substrate can be patterned and used for sample detection, either entirely or partially, and can be used to detect the same or different samples, depending on the applied regeneration process. Figure 21 shows an exemplary workflow for surface regeneration of a patterned substrate formed by any of the techniques disclosed in this invention. Substrate fabrication (block 1200) comprises patterning the upper layer of the substrate (Figures 14-16) to expose or reveal the bonding sites or bonding layers. Activation (block 1202) comprises generating two different reactive groups on the bonding sites and the intermediate or remaining upper layer, for example, a metal oxide layer. This generation can be achieved using plasma treatment or other chemical treatment. Passivation of interstitial regions (block 1204) comprises depositing a polymer limited to the interstitial planes while leaving the bonding sites unmodified.

[0129] DNA origami arrangement (block 1206) involves loading DNA origami (e.g., core structure 13 provided herein) at the binding site, which can carry one or more specific anchor molecules that can anchor other molecular cargo on top of it. These specific anchor molecules may be single-strand DNA, thiols, amines, azides, DBCOs, etc. Cargo loading (block 1208) is a process that allows loading one or more specific molecules onto the planar-organized DNA origami, for example, through links to 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 specimens in a sample. In some embodiments, the specific molecules may comprise a capture molecule and a detector molecule. An assay is performed to evaluate binding on the DNA origami to quantify the number of specimens in the sample (step 1210). Sample storage (block 1212) involves the optional step of storing the substrate after the assay has been performed on it.

[0130] The first regeneration method involves the removal of cargo after assay (block 1230). An exemplary cargo removal protocol is shown in Figures 22-23. The second regeneration method involves the removal of the DNA origami itself using the exemplary procedure shown in Figure 24 (block 1240). 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 types.

[0131] Figure 22 illustrates an exemplary cargo removal procedure 1230, similar to that in Figure 21. This procedure can be performed or initiated using a substrate such as a patterned substrate formed as shown in Figures 14-17, in step 1300. In the illustrated example, the substrate has unloaded DNA origami associated with each binding site on the binding layer. Step 1302 shows the cargo being left at a constant temperature on a substrate having DNA origami, which allows for the loading of single cargo elements onto the DNA origami using specific anchor sites as docking sites. The cargo can be a single molecule or a group of molecules. It is conceivable that a single cargo or multiple molecules can be present on each DNA origami. Step 1304 shows the addition of assay components that interact with the cargo and consequently generate specific signals. After the assay is performed, in step 1306, specific separation molecules are added to the substrate to separate the cargo molecules from the DNA origami, resulting in a substrate in step 1308 that returns to step 1300 with the unloaded DNA origami associated with the binding sites.

[0132] Figure 23 illustrates an exemplary cargo removal procedure 1230 similar to that in Figure 21. This procedure can be performed or initiated in step 1400 using a substrate such as a patterned substrate formed as shown in Figures 14–17. Step 1402 shows the cargo being left at a constant temperature on a substrate having DNA origami, which allows a single cargo element 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 thought that a single cargo or multiple molecules can be present on each DNA origami. Step 1404 shows the addition of assay components that are thought to interact with the cargo and thereby generate a specific signal. After the assay is performed, in step 1406, specific ssDNA is added to the solution that is thought to interact with the cargo molecules and separate the cargo molecules by a process known as DNA strand substitution, resulting in a substrate similar to the one in step 1400, with the unloaded DNA origami associated with the binding sites, which is then returned in step 1408.

[0133] Figure 24 illustrates an exemplary cargo removal procedure 1240 similar to that in Figure 21. This procedure can be performed or initiated using a substrate such as a patterned substrate formed as shown in Figures 14–17, in step 1500. Step 1502 shows the cargo being left at constant temperature on a substrate having DNA origami, which allows single cargo elements to be loaded onto the DNA origami using specific anchor sites as docking sites. The cargo can be a single molecule or a group of molecules. It is thought that a single cargo or multiple molecules can be present on each DNA origami. Step 1504 shows the addition of assay components that are thought to interact with the cargo and consequently generate specific signals. After the assay has been performed, in step 1506 the substrate is treated with a highly reactive solution, which can be a strong acid (pH<2) or a strong base (pH>10), which is thought to be usable to remove all organic molecules, including the cargo molecules, DNA origami, and passivation polymer layer, from the substrate. Subsequently, the method, for example, performs the steps shown in Figures 14 to 17 to form a substrate patterned with 1508 and generate a surface to which single origami is bonded 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 illustrated and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Those skilled in the art will conceive of numerous variations, changes, and substitutions without departing from the present invention. It should be understood that various substitutes for the embodiments of the present invention described herein can be employed to carry out the present invention. The following claims define the scope of the present invention, and the methods and structures contained within these claims and their equivalents are intended to be exhausted therefrom. [Explanation of Symbols]

[0135] 40 Supramolecular structures 504 Bonding layer 506 Upper layer 532 Non-Dynamic Layer 542 Joint area 550 base plate

Claims

1. A method for detecting sample molecules present in a sample using a substrate, A step of providing the substrate having a plurality of binding sites, wherein each of the plurality of binding sites is associated with a corresponding supramolecular structure among the plurality of supramolecular structures, A core structure comprising multiple core molecules, A capture molecule linked to the core structure at the first location, A detector molecule linked to the core structure at a second location, wherein the supramolecular structure is in an unstable state, thereby releasing the detector molecule from the core structure at the second location through the severance of the link between them, The aforementioned step of providing, The step of bringing the sample into contact with the supramolecular structure, thereby shifting the supramolecular structure from the unstable state to the stable state, and linking the detector molecule and the capture molecule to each other through binding to the sample molecule, thereby forming a link between the detector molecule and the capture molecule, and further comprising each of the supramolecular structures in the unstable state, separated by a predetermined distance, A step of providing a deconstruction molecule to trigger the breaking of the link between the detector molecule and the core structure at the second location, wherein the detector molecule remains linked to the core structure through the link with the capture molecule, and the sample molecule becomes associated with the individual binding sites, A step of detecting the sample molecule based on the signal provided by the supramolecular structure that has shifted to the stable state, A method characterized by comprising:

2. The method according to claim 1, characterized in that each of the plurality of binding sites is associated with each of the plurality of supramolecular structures.

3. The method according to claim 1, characterized in that each of the multiple binding sites is associated with each of the multiple single supramolecular structures such that no single binding site is associated with more than one supramolecular structure.

4. The method according to claim 1, characterized in that the supramolecular structure is associated with the individual bonding sites through one or more surface groups present on the bonding layer of the substrate.

5. The method according to 4, characterized in that the core structure of the supramolecular structure forms salt bridges with the binding layer of the substrate at each of the binding sites.

6. The method according to 4, characterized in that 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. Each of the aforementioned bonding portions has x and y dimensions extending across the upper 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. The method according to feature 1.

8. The method according to claim 1, characterized in that the substrate comprises an upper layer patterned to expose the plurality of bonding sites on or within the bonding layer.

9. The method according to 8, characterized in that the binding layer comprises silicon, silicon dioxide, silicon nitride, graphene, quartz, gold, silver, metal, platinum, palladium, PDMS, or a polymer film.

10. The upper layer consists of a metal oxide, graphene, and HfO ２ , or CO ２ The method according to 8, characterized by comprising:

11. The method according to 8, characterized in that the bonding layer is plasma-treated or chemically treated to generate reactive surface groups.

12. The method according to 8, characterized in that the substrate comprises a passivation polymer disposed on the upper layer and not disposed on the plurality of bonding sites.

13. The method according to 8, characterized in that the substrate is planar.

14. The substrate comprises a plurality of wells, The plurality of binding sites are distributed in each of the wells among the plurality of wells. The method according to feature 1.

15. The method according to claim 1, further comprising the step of quantifying the concentration of the sample molecule in the sample.

16. The method according to claim 1, further comprising the step of identifying the detected sample molecule.

17. The method according to claim 1, further comprising the step of detecting the sample molecule based on the signal when the sample molecule is present in the sample as a single molecule or in a higher-order number.

18. The aforementioned sample comprises a composite biological sample, The method provides single-molecule sensitivity, thereby increasing the dynamic range and quantitative capture of various molecular concentrations within the composite biological sample. The method according to feature 1.

19. The method according to claim 1, characterized in that the sample molecule comprises a protein, peptide, peptide fragment, lipid, DNA, RNA, organic molecule, inorganic molecule, or any combination thereof.

20. The method according to claim 1, characterized in that each of the plurality of supramolecular structures is a nanostructure.

21. The method according to claim 1, characterized in that the core structure of each of the supramolecular structures is a nanostructure.

22. The method according to claim 1, characterized in that the plurality of core molecules of the core structure are arranged in a predetermined shape and / or have a specified molecular weight.

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