KR20230148417A - Structures and methods for detection of sample analytes - Google Patents

Abstract

Provided herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, one or more analyte molecules are detected using one or more supramolecular structures. In some embodiments, supramolecular structures facilitate binding of single detector molecules. In some embodiments, the supramolecular structure in the steady state is configured to provide a signal for analyte molecule detection and quantification. In some embodiments, the signal relates to a DNA signal such that detection and quantification of the analyte molecule includes converting the presence of the analyte molecule into a DNA signal.

Description

Structures and methods for detection of sample analytes

Cross-reference to related applications

This application claims priority and benefit to U.S. Provisional Application No. 63/152,607, filed February 23, 2021 and entitled “STRUCTURE AND METHODS FOR DETECTION OF SAMPLE ANALYTES,” the disclosure of which is hereby incorporated by reference in its entirety. incorporated by reference.

The current state of personalized health care is overwhelmingly genome-centric, focusing primarily on quantifying the genes present within an individual. Although these approaches have proven to be very powerful, they do not provide clinicians with a complete picture of an individual's health. Genes are an individual's "blueprint" and only indicate the likelihood of developing a disease. In order for these "blueprints" within an individual to have any effect on the individual's health, they first need to be transcribed into RNA and then translated into various protein molecules that are the actual "factors" in the cell.

Protein concentrations, interactions between proteins (protein-protein interactions or PPIs), as well as interactions between proteins and other molecules are complex with the health and homeostasis regulation mechanisms of various organs, as well as the interaction of these systems with the external environment. It is related. Therefore, quantitative information about proteins and protein interactions, such as PPIs, is essential not only to generate a complete picture of an individual's health at a given point in time, but also to predict any new health problems. For example, the amount of stress experienced by the heart muscle (e.g. during a heart attack) can be inferred by measuring the concentrations of troponin I/II and myosin light chains present in peripheral blood. Similar protein biomarkers have also been identified for a wide variety of organ dysfunctions (e.g. liver disease and thyroid disorders), certain cancers (e.g. colorectal or prostate cancer), and infectious diseases (e.g. HIV and Zika). , verified and utilized. Interactions between these proteins are also essential for drug development and are becoming an increasingly highly demanded dataset. The ability to detect and quantify proteins and their interactions with other molecules within a given body fluid sample is an essential element of these healthcare developments.

summary

The present disclosure generally relates to systems, structures, and methods for detection and quantification of analyte molecules in a sample.

In some embodiments, a) providing a supramolecular structure comprising a core structure comprising a plurality of core molecules and a capture molecule connected to the core structure at a first position, b) contacting the sample with the supramolecular structure, and c) providing a detector molecular assembly; and d) detecting the analyte molecule based on the signal provided by the detector molecular assembly and the associated signal provided by the supramolecular structure. Provided herein is a method of detecting an analyte molecule present in a sample.

In some embodiments, any of the methods disclosed herein further comprise quantifying the concentration of analyte molecules in the sample. In some embodiments, any of the methods disclosed herein further comprise identifying the detected analyte molecule. In some embodiments, any of the methods disclosed herein further comprise detecting an analyte molecule based on a signal when the analyte molecule is present in the sample in counts of more than a single molecule. In some embodiments, for any of the methods disclosed herein, the sample comprises a complex biological sample, and the method provides single-molecule sensitivity, thereby increasing the dynamic range and quantitative capture of a wide range of molecular concentrations within the complex biological sample. . In some embodiments, for any of the methods disclosed herein, the analyte 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.

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

In some embodiments, for any of the methods disclosed herein, each analyte molecule is 1) bound to a capture molecule of the respective supramolecular structure via a chemical bond, and/or 2) bound to a detector molecular assembly via a chemical bond. do. In some embodiments, for any of the methods disclosed herein, the capture molecule and detector molecule are independently a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, darpin, catalyst, polymerization initiator, polymer such as PEG. , or a combination thereof. In some embodiments, for any of the methods disclosed herein, for each supramolecular structure: a) a capture molecule is linked to the core structure via a capture barcode, wherein the capture barcode is a first capture linker, a second capture linker, and a capture bridge disposed between the first capture linker and the second capture linker, wherein the first capture linker is bound to the first core linker bound to the first position on the core structure, and wherein the capture molecule and the second capture linker. The linkers are linked together via linkage to a third capture linker, and b) the detector molecule assembly comprises a detector barcode, wherein the detector barcode comprises one or more linkers. In some embodiments, the capture crosslink and detector molecular assemblies independently comprise a polymer core. In some embodiments, the polymeric core of the capture crosslink and the polymeric core of the detector molecular assembly independently comprise a nucleic acid (DNA or RNA) of a specific sequence or a polymer, such as PEG.

In some embodiments, for any of the methods disclosed herein, each supramolecular structure further comprises an anchor molecule connected to the core structure. In some embodiments, the anchor molecule is linked to the core structure via an anchor barcode, wherein the anchor barcode comprises a first anchor linker, a second anchor linker, and an anchor bridge disposed between the first anchor linker and the second anchor linker. And, the first anchor linker is linked to the third core linker linked to the third position on the core structure, and the anchor molecule is linked to the second anchor linker. In some embodiments, the anchor molecule is one or more molecules such as an amine, thiol, DBCO, maleimide, biotin, azide, acrydite, NHS-ester, a single-stranded nucleic acid (RNA or DNA) of a specific sequence, a polymerization initiator, or PEG. Including polymers, or combinations thereof. In some embodiments, the anchor crosslink comprises a polymer core. In some embodiments, the polymeric core of the anchor bridge comprises a specific sequence of nucleic acid (DNA or RNA) or a polymer such as PEG. In some embodiments, the third core linker, first anchor linker, second anchor linker and anchor molecule independently comprise an anchor reactive molecule or DNA sequence domain. In some embodiments, each anchor-reactive molecule is independently an amine, thiol, DBCO, maleimide, biotin, azide, acrydite, NHS-ester, a single-stranded nucleic acid (RNA or DNA) of a specific sequence, a polymerization initiator, or and one or more polymers, such as PEG, or combinations thereof. In some embodiments, the anchor molecule is linked to a second anchor linker through a chemical bond. In some embodiments, the anchor molecule is covalently linked to a second anchor linker.

In some embodiments, for any of the methods disclosed herein, the signal comprises a detector barcode, capture barcode, or combination thereof corresponding to a supramolecular structure that is bound to the analyte and then bound back to the detector molecular assembly. In some embodiments, each detector barcode provides a DNA signal corresponding to the detector molecule, providing information indicating its specificity for the analyte molecule bound to each detector molecule. In some embodiments, the detector barcode is analyzed using genotyping, qPCR, sequencing, or a combination thereof. In some embodiments, multiple analyte molecules in a sample are detected simultaneously through multiplexing. In some embodiments, for any of the methods disclosed herein, the capture molecule and detector molecule for each supramolecular structure are configured to bind one or more specific types of analyte molecules.

In some embodiments, for any method disclosed herein comprising using a plurality of supramolecular structures, each core structure of the plurality of supramolecular structures is identical to each other. In some embodiments, each supramolecular structure comprises a defined shape, size, molecular weight, or combination thereof to reduce or eliminate cross-reaction between the plurality of supramolecular structures. In some embodiments, each supramolecular structure comprises a plurality of capture molecule molecules. In some embodiments, each supramolecular structure comprises capture molecules and detector molecules of defined stoichiometry to reduce or eliminate cross-reaction between the plurality of supramolecular structures.

In some embodiments, one or more supramolecular structures are attached to a hydrogel porous matrix. In some embodiments, each supramolecular structure is copolymerized with a hydrogel via a corresponding anchor molecule linked to each core structure of the corresponding supramolecular structure. In some embodiments, one or more supramolecular structures are embedded within the hydrogel. In some embodiments, a plurality of supramolecular structures are disposed on a substrate, such as a shaped or planar substrate, wherein the substrate comprises a plurality of binding sites, each binding site being configured to connect with a corresponding supramolecular structure. In some embodiments, multiple supramolecular structures are configured to detect the same analyte molecule. In some embodiments, for any method that includes using a substrate, the method further comprises providing a plurality of signaling elements coupled to a detector molecule. In some embodiments, each signaling element comprises a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle or polymer. In some embodiments, at least one supramolecular structure in the plurality of supramolecular structures is configured to detect a different analyte molecule than the other supramolecular structures.

In some embodiments, for any method that includes using a planar substrate, the method further includes barcoding each supramolecular structure to identify the location of each supramolecular structure on the planar substrate. In some embodiments, for any method that includes using a planar substrate, the method includes providing a plurality of signaling elements as provided herein configured to couple with a detector molecule.

In some embodiments, for any of the methods disclosed herein, the sample comprises a biological particle or biomolecule. In some embodiments, for any of the methods disclosed herein, the sample is an aqueous solution comprising a protein, peptide, fragment of a peptide, lipid, DNA, RNA, organic molecule, viral particle, exosome, organelle, or any complex thereof. Includes. In some embodiments, for any of the methods disclosed herein, a sample may include tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture medium, discarded tissue, plant material, synthetic protein, prion, bacterial and/or viral samples or fungal tissue, or combinations thereof. The sample may be processed to release the analyte from the cells or otherwise prepare the sample for analysis prior to contacting the sample with the supramolecular structures provided herein. The sample may be an environmental sample, such as wastewater or soil sample. The sample may also be a non-biological sample. In one embodiment, the sample may be a sample from a chemical processing step, a sample of a food or nutritional ingredient, or a packaging ingredient.

In some embodiments, provided herein is a substrate for detecting one or more analyte molecules in a sample, comprising a plurality of supramolecular structures, wherein each supramolecular structure includes a) a core structure comprising a plurality of core molecules, and b) Contains a capture molecule linked to the supramolecular core.

In some embodiments, each core structure of the plurality of supramolecular structures is identical to each other. In some embodiments, the substrate comprises a solid support, solid substrate, polymer matrix, or molecular condensate. In some embodiments, the sample comprises a complex biological sample, and the method provides single-molecule sensitivity, thereby increasing the dynamic range and quantitative capture of a wide range of molecular concentrations within the complex biological sample. In some embodiments, the one or more analyte molecules comprise a protein, peptide, peptide fragment, lipid, DNA, RNA, organic molecule, inorganic molecule, complex thereof, or any combination thereof. In some embodiments, each supramolecular structure is a nanostructure. In some embodiments, each core structure is a nanostructure. In some embodiments, the plurality of core molecules for each core structure are arranged in a predefined shape and/or have a defined molecular weight. In some embodiments, the predefined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. In some embodiments, the plurality of core molecules for each core structure comprises one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, each core structure is independently a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tile structure, It includes multi-stranded DNA tile structures, single-stranded RNA origami, multi-stranded RNA tile structures, hierarchically organized DNA or RNA origami with multiple scaffolds, peptide structures, or combinations thereof. In some embodiments, each analyte molecule is 1) bound to a capture molecule via a chemical bond and/or 2) bound to a detector molecule via a chemical bond. In some embodiments, the capture molecule and detector molecule independently include a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, darpin, catalyst, polymerization initiator, polymer such as PEG, or combinations thereof.

In some embodiments, for each supramolecular structure of the substrate: a) a capture molecule is linked to the core structure via a capture barcode, wherein the capture barcode includes a first capture linker, a second capture linker, and a first capture linker and a second capture linker. a capture bridge disposed between two capture linkers, wherein the first capture linker is bound to the first core linker bound to a first position on the core structure, and wherein the capture molecule and the second capture linker are attached to the third capture linker. are connected together through bonding. The capture molecule binds to the analyte, which in turn binds to the detector molecule of the detector molecule assembly. In one embodiment, the detector molecular assembly comprises separate supramolecular structures that are not linked to a capture molecule and are not anchored on a substrate.

In some embodiments, the supramolecular structure comprising a capture molecule interacts directly with the substrate material to immobilize the supramolecular structure on the substrate. In some embodiments, the supramolecular structure comprising a capture molecule further comprises an anchor molecule as provided herein linked to the core structure, wherein the anchor molecule is linked to the substrate to anchor the supramolecular structure on the substrate.

In some embodiments, the signal read or detected from the substrate includes a detector barcode, a capture barcode, or a combination thereof that can be analyzed using optical sensors, magnetic sensors, and/or electrical sensors. Detection techniques include electrochemical detection, genotyping, qPCR, sequencing, or combinations thereof. In some embodiments, one or more supramolecular structures are configured to multiplex a sample, where multiple analyte molecules within the sample are detected simultaneously. In some embodiments, the capture molecule for each supramolecular structure is configured to bind one or more specific types of analyte molecules.

In some embodiments, the sample comprises a complex biological sample, and the method provides single-molecule sensitivity, thereby increasing the dynamic range and quantitative capture of a wide range of molecular concentrations within the complex biological sample. In some embodiments, the analyte molecule comprises a protein, peptide, peptide fragment, lipid, DNA, RNA, organic molecule, inorganic molecule, complex thereof, or any combination thereof. In some embodiments, the supramolecular structure is a nanostructure. In some embodiments, the core structure is a nanostructure. In some embodiments, the supramolecular structure comprises a defined shape, size, molecular weight, or combination thereof to reduce or eliminate cross-reaction with another supramolecular structure. In some embodiments, the supramolecular structure includes a plurality of capture molecules.

In some embodiments, the plurality of core molecules for a core structure are arranged in a predefined shape and/or have a defined molecular weight. In some embodiments, the predefined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. In some embodiments, the plurality of core molecules for each core structure comprises one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, the core structure is an independently scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tile structure, a multi- stranded DNA tile structures, single-stranded RNA origami, multi-stranded RNA tile structures, hierarchically organized DNA or RNA origami with multiple scaffolds, peptide structures, or combinations thereof.

Specific embodiments of the disclosed devices, delivery systems, or methods will now be described with reference to the drawings. Nothing in this detailed description is intended to imply that any particular component, feature or step is essential to the invention.
1 shows a supramolecular structure and related subcomponents according to an embodiment of the present disclosure.
Figure 2 shows a supramolecular structure with associated analyte and detector molecular assemblies according to an embodiment of the present disclosure.
Figure 3 shows the supramolecular structure of Figure 1, with associated analyte and detector molecular assemblies and associated subcomponents according to embodiments of the present disclosure.
Figure 4 shows a detector molecular assembly comprising a detector core structure and a supramolecular structure with associated analyte according to an embodiment of the present disclosure.
Figure 5 shows specific binding of supramolecular structures and detector molecular assemblies to analytes according to embodiments of the present disclosure.
Figure 6 shows an analyte without specific binding to a supramolecular structure according to an embodiment of the present disclosure.
Figure 7 shows a workflow for analyte and detector coupling to an array of supramolecular structures according to an embodiment of the present disclosure.
8 shows exemplary capture molecule identification used with analyte detection according to embodiments of the present disclosure.
Figure 9 shows an example of analyte detection according to an embodiment of the present disclosure.
Figure 10 shows an example of analyte detection according to an embodiment of the present disclosure.
Figure 11 shows an example of an array of supramolecular structures that can be used for analyte detection according to embodiments of the present disclosure.
Figure 12 shows an example of an array of supramolecular structures that can be used for analyte detection according to embodiments of the present disclosure.
Figure 13 shows an example of an array of supramolecular structures that can be used for analyte detection according to embodiments of the present disclosure.
Figure 14 shows an example of an array of supramolecular structures that can be used for antigen detection according to embodiments of the present disclosure.
Figure 15 shows an exemplary porous matrix supramolecular structure array that can be used for analyte detection according to embodiments of the present disclosure.
Figure 16 shows a block diagram of an exemplary analyte detection system according to embodiments of the present disclosure.

details

Disclosed herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, one or more analyte molecules are detected based on capture by one or more supramolecular structures. In some embodiments, one or more supramolecular structures comprise or are linked to a capture molecule that specifically binds to an analyte present in the sample. The bound analyte can in turn be identified by a detector molecule (e.g., Contains, interacts with, or interacts with other molecules that can be detected (optically, electrically, magnetically), or can be used to dock them. In some embodiments, the detector molecular assembly comprises a DNA signal such that detection and quantification of binding of an analyte molecule to a capture molecule comprises converting the presence of the analyte molecule into a DNA signal through amplification of a unique identifier of the supramolecular structure. creates . In some embodiments, the detector molecular assembly is linked to an enzyme that converts the substrate into an optically detectable signal. In one embodiment, the supramolecular structure is a nucleic acid origami linked to or immobilized on a substrate. In one embodiment, the supramolecular structure retains the capture molecule via a barcode that includes a unique identifier for the capture molecule and connects the capture molecule to the scaffold of the supramolecular structure.

In some embodiments, the disclosed technology provides a single molecule enzyme-linked immunosorbent assay (ELISA), wherein a supramolecular structure and an associated capture molecule act as a capture entity, and a detector molecule assembly is used to generate a detection signal. Acts as a detection entity (e.g., when reacting with an appropriate detection reagent that is detected using a sensor in a detection system). The use of supramolecular structures as capture entities allows specific identification and, in some embodiments, position mapping of each individual capture molecule immobilized on a substrate. Additionally, supramolecular structures are configured to be organized on a substrate or within a porous material to allow single molecule bonding.

Accordingly, as provided herein, detectable analyte binding can be associated with an individual capture molecule among many different capture molecules on a substrate, producing an assay result in which the binding properties of an analyte pool of multiple different analytes are characterized. . This ultimately allows samples with analytes of uncharacterized composition to be analyzed for the presence and/or concentration of the specific analyte of interest. For example, a human sample can be characterized to determine the presence and/or concentration of antibodies with binding specificity for a particular antigen within a panel of antigen capture molecules, thereby allowing the capture molecules to match a panel of known infectious disease antigens. indicates. The assay result may indicate a positive binding result associated with a particular antigen, indicating the presence of antibodies in the subject providing the sample. In another embodiment, the identity of the analyte in the sample may be at least partially known, but its binding affinity may not be characterized for a particular pool of capture molecules. For example, the capture molecule can be a set of candidate drugs and the analyte can be a molecule in human blood. Binding of drug candidates to these proteins can be used to assess bioavailability or potential off-target binding. Assay results are based on the identification of a specific detector binding (e.g., identifying the binding by barcode identification on a detector molecular assembly containing an antibody specific for the analyte), which can be mapped to a specific analyte. It may indicate a positive binding result associated with the drug candidate.

While conventional ELISA protocols may include a detectable fluorescent signal produced by an enzyme coupled to a detection antibody as an indicator of binding, the disclosed technology additionally or alternatively provides an amplified nucleic acid signal from a unique identifier of the detector molecular assembly. sequence information can be determined from which or an optically detectable signal corresponding to amplification (e.g., qPCR using a set of primers/probes specific for the unique identifier) is emitted. Accordingly, unique identifying information for an assembly of detector molecules allows, in certain embodiments, specific identification of a particular detector molecule linked to a capture molecule via a bound analyte. However, in embodiments, the detector molecular assembly may not possess a unique identifier. Other detection techniques may include optical, magnetic and/or electrical detection techniques.

Sample

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

Supramolecular structures

In some embodiments, supramolecular structures are programmable structures that can spatially organize molecules. In some embodiments, a supramolecular structure comprises a plurality of molecules linked together. In some embodiments, the plurality of molecules of the supramolecular structure interact with at least a portion of each other. In some embodiments, the supramolecular structure comprises a specific shape. In some embodiments, the supramolecular nanostructure comprises a defined molecular weight based on the plurality of molecules of the supramolecular structure. In some embodiments, the supramolecular structure is a nanostructure. In some embodiments, a plurality of molecules are linked together through bonds, chemical bonds, physical attachments, or combinations thereof. In some embodiments, supramolecular structures comprise large molecular entities of specific shapes and molecular weights formed from a well-defined number of smaller molecules that specifically interact with each other. In some embodiments, the structural, chemical and physical properties of the supramolecular structures are explicitly designed. In some embodiments, the supramolecular structure includes a plurality of subcomponents spaced apart by a defined distance. In some embodiments, at least a portion of the supramolecular structure is rigid. In some embodiments, at least a portion of the supramolecular structure is semi-rigid. In some embodiments, at least a portion of the supramolecular structure is flexible.

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

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

In one embodiment, the nucleic acid origami has at least one lateral dimension from about 50 nm to about 1 μ. In one embodiment, the nucleic acid origami is, for example, from about 50 nm to about 200 nm, from about 50 nm to about 400 nm, from about 50 nm to about 600 nm, from about 50 nm to about 800 nm, from about 100 nm to about 200 nm, It has at least one lateral dimension of about 100 nm to about 300 nm, about 100 nm to about 400 nm, about 100 nm to about 500 nm, about 200 nm to about 400 nm. In one embodiment, the nucleic acid origami has at least a first lateral dimension of about 50 nm to about 1 μ and a second lateral dimension orthogonal to the first lateral dimension of about 50 nm to about 1 μ. In one embodiment, the nucleic acid origami has a planar footprint of about 200 nm ² to about 1 μ ² in area.

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

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

In some embodiments, a particular first core linker 12 is disposed at a first location on the core structure. In some embodiments, one or more core molecules at the first position are modified to form a linkage with the first core linker (12). In some embodiments, first core linker 12 is an extension of core structure 13.

In some embodiments, a particular third core linker (14) is disposed at a third position on the core structure (13). In some embodiments, one or more core molecules at the third position are modified to form a linkage with a third core linker (14). In some embodiments, third core linker 14 is an extension of core structure 13. In some embodiments, the first location and the second location 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.

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

In some embodiments, each component of the supramolecular structure can be independently modified or adjusted. In some embodiments, modifying one or more of the components of the supramolecular structure may modify the 2D and 3D geometry of the supramolecular structure itself. In some embodiments, modifying one or more of the components of the supramolecular structure can modify the 2D and 3D geometry of the core structure. In some embodiments, this ability to independently modify components of a supramolecular nanostructure is achieved by forming one or more supramolecular structures on a solid surface (e.g., a planar surface or microparticle) and a 3D volume (e.g., within a hydrogel matrix). It enables precise control over the organization of

capture barcode

As shown in Figure 1, in some embodiments, capture molecule 2 is linked to core structure 13 via capture barcode 20. In some embodiments, capture barcode 20 forms a link with capture molecule 2 and capture barcode 20 forms a link with core structure 13. In some embodiments, capture barcode 20 includes a first capture linker 11, a second capture linker 6, and a capture bridge 7. In some embodiments, the first capture linker 11 comprises a reactive molecule. In some embodiments, the first capture linker 11 is an amine, thiol, DBCO, NHS ester, maleimide, azide, acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer. (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the first capture linker 11 comprises a DNA sequence domain. In some embodiments, the second capture linker 6 comprises a reactive molecule. In some embodiments, the second capture linker 6 is an amine, thiol, DBCO, NHS ester, biotin, maleimide, azide, acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a reactive molecule comprising a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second capture linker comprises a DNA sequence domain. In some embodiments, capture crosslink 7 comprises a polymer. In some embodiments, the capture crosslink 7 comprises a polymer comprising a nucleic acid (e.g., DNA or RNA) of a specific sequence. In some embodiments, the capture crosslink (7) comprises a polymer, such as PEG. In some embodiments, the first capture linker (11) is attached to the first distal end of the capture bridge (7) and the second capture linker (6) is attached to the second distal end of the capture bridge (7). In some embodiments, the first capture linker (11) is attached to the capture bridge (7) via a chemical bond. In some embodiments, the second capture linker (6) is attached to the capture bridge (7) via a chemical bond. In some embodiments, the first capture linker (11) is attached to the capture bridge (7) via physical attachment. In some embodiments, the second capture linker (6) is attached to the capture bridge (7) via physical attachment.

In some embodiments, capture barcode 20 is connected to core structure 13 via a connection between first capture linker 11 and first core linker 12. In some embodiments, as described herein, the first core linker 12 is disposed at a first location on the core structure 13. In some embodiments, first capture linker 11 and first core linker 12 are linked together via chemical bonds. In some embodiments, the first capture linker 11 and the first core linker 12 are linked together via a covalent bond.

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

anchor barcode

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

In some embodiments, the anchor barcode is connected to the core structure (13) through a connection between the first anchor linker (15) and the third core linker (14). In some embodiments, as described herein, the third core linker 14 is disposed at a third location on the core structure 13. In some embodiments, the first anchor linker 15 and the third core linker 14 are linked together through chemical bonds. In some embodiments, the first anchor linker 15 and the third core linker 14 are linked together via a covalent bond.

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

Figure 2 is a schematic diagram of the supramolecular structure 40 bound to the corresponding analyte molecule 44 with binding specificity for the capture molecule 2. Supramolecular structure 40 may bind to one or more analyte molecules 44 depending on the specific capture molecule 2 associated with supramolecular structure 40. Analyte molecule 44 may also bind to detector molecule assembly 46. The detector molecule assembly 46 includes a detector molecule 1 that specifically binds to an analyte molecule 44. Figure 2 shows a sandwich-like binding arrangement in which a capture molecule (2) and a detector molecule (1) bind to different sites on the analyte molecule (44). In some embodiments, detector molecule 1 is a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, nanobody, darpin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or combinations thereof. Includes. As shown in Figure 2, in some embodiments, detector molecule 1 is linked to detector barcode 21. In some embodiments, detector barcode 21 forms a link with detector molecule 1 and may include one or more intervening components. In other embodiments, detector molecule 1 is linked to a detectable tail or linker, but does not possess unique barcode information.

3 shows an exemplary arrangement of detector barcodes 21. In some embodiments, the detector barcode comprises one or more detector linkers comprising a first detector linker (4). The detector barcode 21 may include a dock 8 that functions as an attachment or extension/amplification site to facilitate detection. In some embodiments, linker 4 comprises a reactive molecule. In some embodiments, linker 4 is an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer ( reactive molecules including, for example, polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, linker 4 comprises a DNA sequence domain. In some embodiments, dock 8 comprises a polymer. In some embodiments, dock 8 comprises a polymer comprising a nucleic acid (DNA or RNA) of a specific sequence, eg, a single-stranded or double-stranded nucleic acid. In some embodiments, dock 8 comprises a polymer, such as PEG. In some embodiments, linker 4 is attached to dock 8 at its distal end and another detector linker 4 is attached to dock 8 at its second distal end. Attachment may be through chemical bonds or physical attachment.

In some embodiments, detector barcode 21 is attached to detector molecule 1 via a linkage between a plurality of linkers presented herein as detector linker 4 and a second detector linker 3 linked to detector molecule 1. connected. In some embodiments, the second detector linker 3 comprises a reactive molecule. In some embodiments, the second detector linker 3 is an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a reactive molecule comprising a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second detector linker 3 comprises a DNA sequence domain. In some embodiments, the detector molecule (1) is coupled to the second detector linker (3) via a chemical bond. In some embodiments, detector molecule (1) is coupled to a second detector linker (3) via a covalent bond. In some embodiments, second detector linker 4 and second detector linker 3 are linked together via a chemical bond. In some embodiments, second detector linker 4 and second detector linker 3 are linked together via a covalent bond.

As provided herein, the capture molecule assembly includes a supramolecular structure (40) having a core structure (13) and a capture molecule (1). In certain embodiments, detector molecular assembly 46 is connected or coupled to core structure 13, such that the detector molecular assembly is also a supramolecular structure provided herein. Accordingly, as shown in Figure 4, the binding of the detector molecule to the captured analyte molecule 44 associated with the capture molecule assembly supramolecular structure 40 creates a supramolecular structure sandwich. Typically, only one of the detector molecular assembly 46 or the supramolecular structure 40 of the capture molecular assembly is fixed to allow flow of the capture or detection entity. In one embodiment, the capture molecule assembly is anchored within or on a surface of a porous material.

Supramolecular structure 40 and/or detector molecular assembly 46 may include DNA origami. In some embodiments, subcomponents of the core structure 13 of the supramolecular structure 40 and/or detector molecular assembly 46 include DNA origami as well as one or more extended nucleic acid strands. In some embodiments, the core structure 13 of the supramolecular structure 40 and/or the detector molecular assembly 46 comprises a scaffolded DNA origami, wherein circular ssDNA molecules, referred to as “scaffold” strands, are called ssDNA “ It is folded into a predefined 2D or 3D shape by interacting with two or more short ssDNAs, called "staple" strands, which interact with specific sub-sections of the "scaffold" strand.

As described herein, in some embodiments, one or more supramolecular structures enable detection of one or more analyte molecules in a sample. As shown in the schematic diagram of Figure 5, supramolecular structure 40 with associated capture molecules is exposed to analyte molecules 44 and detector molecular assembly 46. If the individual analyte molecule 44 and the individual capture molecule 2 have binding specificity for each other, the analyte molecule 44 associates with the capture molecule 2. Again, the detector molecule assembly 46, which has binding specificity for the analyte molecule 44, associates with the analyte molecule 44 to create the bound detection structure 50.

As shown in Figure 6, the analyte molecule 44 that does not have binding specificity for the capture molecule 2 does not associate with the supramolecular structure 40. As a result, the detector molecular assembly 46 also does not associate with the supramolecular structure 40, and the combined detection structure 50 is not created. For a substrate comprising an immobilized array of supramolecular structures 40, if the sample contains an appropriate analyte molecule 44 with binding specificity for the capture molecule 2, each individual site is bound to the detection structure. It can have a coupling reaction to produce (50).

Although the depicted embodiment shows detector molecule assembly 46 and analyte molecule 44 in contact with supramolecular structure 40 in one step, analyte molecule 44 and detector molecule assembly 46 are not described herein. It should be understood that the addition may be in a separate step as provided, and that any unbound analyte molecules 44 are removed prior to addition to the detector molecular assembly 46. Figure 7 shows an exemplary method workflow for adding analyte molecules 44 to a pool or array of capture molecule assemblies implemented as supramolecular structures 40. Analyte molecules 44 may represent different analytes present in the sample, such that different analyte molecules 44 in the pool have different degrees of binding specificity to the array of available capture molecules 2.

Reaction conditions allow analyte molecules 44 to bind to specific capture molecules 2. As provided herein, binding specificity may refer to the interaction between the capture molecule 2 and the analyte molecule 44 that remains intact under reaction conditions and after washing or removal steps for unbound reagents. Binding specificity may include the formation of covalent or non-covalent bonds, ionic bonds, dipolar interactions, hydrophilic or hydrophobic interactions, complementary nucleic acid bonds, etc. Specific binding may mean binding to an analyte molecule (44) that binds only to a specific capture molecule (2) and not to other capture molecules (2). Thus, certain capture molecules 2 of the array (e.g., capture molecule 2a) bind to analyte molecules 44, while other capture molecules (e.g., capture molecule 2b) bind to a given sample. It has no binding partners available within it and therefore does not bind with specificity to any analyte molecule 44. Any unbound analyte can be removed from the immobilized capture molecule assembly as provided herein.

Detector molecule 1 and analyte molecule 44 may also have binding specificity to each other. The array is then contacted with a detector molecular assembly 46, which may all be the same or different as disclosed in the various embodiments. Any unbound detector molecular assembly 46 is removed, for example by washing. After these workflow steps, a variety of bound detector structures 50, each bound to a respective analyte and detector molecule assembly 46, remain on the array, which provides a variety of detection links that associate the analyte to a specific supramolecular structure identity. The protocol can be applied, which again involves a known capture molecule (2). Therefore, analyte-capture molecule binding is characterized through detection.

8 shows different unique capture barcodes (shown as capture barcodes 20a, 20b, 20c and 20d) of the coupled detector structure 50 to associate a binding event with a specific capture barcode. Example detection steps evaluated are shown. In some embodiments, supramolecular structures convert information regarding the presence of a given analyte molecule in a sample into a DNA signal. In some embodiments, the DNA signal corresponds to sequence data for a capture barcode and/or a detector barcode, where the capture molecule and the detector molecule are simultaneously linked (e.g., bound) to an analyte molecule (e.g., sandwich formation).

In some embodiments, detecting the presence of an analyte molecule as described herein involves introducing single or multiple unique nucleic acid molecules into a solution that is used to identify as well as quantify the properties of the analyte molecule from the sample. Includes controllable release. In some embodiments, the unique nucleic acid molecule is provided by a capture barcode 20 on each supramolecular structure. In some embodiments, detecting the presence of an analyte molecule as described herein includes generating an optical or electrical signal coupled to a change of state that can be counted to quantify the concentration of the analyte molecule in solution.

In some embodiments, multiple analyte molecules are simultaneously detected in a sample through multiplexing, wherein multiple supramolecular structures provide multiple signals (e.g., detector barcodes, capture barcodes) for sequencing and analyte identification. do. In some embodiments, the methods described herein for detecting analytes in a sample provide high throughput and high multiplexing capabilities by using multiple supramolecular structures. In some embodiments, high throughput and high multiplexing capabilities provide high accuracy for analyte molecular detection and quantification. In some embodiments, the methods described herein for detecting analytes in a sample are configured to characterize and/or identify biopolymers, including protein molecules, rapidly and with high sensitivity and reproducibility. In some embodiments, the plurality of supramolecular structures are configured to limit cross-reactivity association errors. In some embodiments, such cross-reactive association errors involve a capture molecule and/or detector molecule of a supramolecular structure interacting with a capture molecule and/or detector molecule of another supramolecular structure (e.g., intermolecular interactions) . In some embodiments, each core structure of the plurality of supramolecular structures is identical to each other. In some embodiments, the structural, chemical and physical properties of each supramolecular structure are explicitly designed. In some embodiments, the same core structure has a defined shape, size, molecular weight, defined number of capture molecules and detector molecules, and a predetermined distance between the corresponding capture molecules and detector molecules to limit cross-reactivity between the supramolecular structures. (as described herein), with a defined stoichiometry between the corresponding capture molecules and detector molecules, or combinations thereof. In some embodiments, the molecular weight of all core structures is the same and accurate to the purity of the core molecules. In some embodiments, each core structure has at least one capture molecule.

In some embodiments, a plurality of supramolecular structures may share structural similarities due to the same specific subcomponents, but the interactions between the analyte molecules from the sample and the supramolecular structures are defined by the corresponding capture and detector molecules. . In some embodiments, each bound detector and capture molecule on a given bound detection structure 50 can specifically interact with a particular analyte molecule in the sample. In some embodiments, each supramolecular structure comprises a unique DNA barcode corresponding to the associated capture molecule. In some embodiments, the capture molecule is designed to interact with more than one analyte molecule in the sample.

As provided herein, capture barcode 20 can be used to uniquely identify individual supramolecular structures 40. Ultimately, each supramolecular structure 40 is assembled such that the capture molecule 2 can be associated, for example, with a capture barcode 20 stored in a lookup table of an analyte detection system (see Figure 16). Accordingly, once the capture barcode 20 is identified, the identity of the capture molecule 2 is also accessible.

In some embodiments, each supramolecular structure is configured for single-molecule sensitivity to ensure the highest possible dynamic range necessary to quantitatively capture the wide range of molecular concentrations within a typical complex biological sample. In some embodiments, multiple supramolecular structures limit or eliminate manipulation of the sample necessary to reduce non-specific interactions as well as any user induced errors.

Figure 9 shows an example analyte detection technique in which different positions of the supramolecular structure 40, each with a different capture molecule 2, can be used to characterize analyte binding along with unique capture barcode information. A sample containing a pool of different analyte molecules (44) is contacted with an immobilized supramolecular structure (40) each having a different capture molecule (2). The analyte molecule 44 and the capture molecule 2, which have binding specificity for each other, are brought into contact under conditions that allow interaction to occur. The detector molecular assembly 46 is allowed to bind to the analyte molecule 44 associated with the supramolecular structure. The coupled detector structure can be characterized based on 1) the capture barcode 20 and 2) the signal produced by the coupled detector molecular assembly corresponding to the location map of the specific capture barcode 20. In one embodiment, the capture barcode information and the location of the supramolecular structure 40 can be determined prior to analyte binding. That is, the array may be provided pre-mapped, or the mapping may be a separate step. Mapping may include detecting capture barcodes as generally provided herein, such as detecting unique optical, electrical and/or magnetic patterns. In one embodiment, detection comprises sequencing the nucleotide sequence of the capture barcode. In one embodiment, detection involves amplification and quantification of the amplified product, e.g., detection of a signal associated with a probe via qPCR.

Serology tests look for antibodies in a patient's blood to confirm previous infection with a pathogen. In one example, a COVID-19 serology assay detects the presence of IgG or IgM antibodies against the spike protein or nucleocapsid. Capture molecule 2 pulls down antibody analyte 44 in the patient sample, which is also bound by detector molecule assembly 46. In one embodiment, the detector molecule assemblies 46 may all be of the same type and/or may all have the same detector molecule 1. Detector molecule 1 may be an anti-human antibody that binds to any human antibody regardless of antibody-antigen specificity. Accordingly, the detector molecule (1) can bind to a variety of different analytes (44) associated with each antigen capture molecule (2). Analytes 44 that exhibit positive binding events through detectable signals from detector molecular assemblies 46 can be linked to specific supramolecular structures 40 based on specific barcodes 20, thereby identifying positive antibody results. there is.

The disclosed technology can be used to generate assays for one or more infectious diseases, such as COVID-19, influenza, RSV, and pneumonia. In one embodiment, the capture molecules 2 comprise a pool of different antigens from different infectious diseases, each associated with a different supramolecular structure 40. Additionally or alternatively, the assay may include multiple isotypes and multiple potential antigens of infectious diseases, as well as antigens from other respiratory pathogens, including but not limited to influenza, RSV, and pneumonia. In one embodiment, the assay may allow for differentiation between natural immunity and acquired immunity from a vaccine through specific addition of vaccine protein targets. Assays may also include distinction between IgG, IgM and IgA specificities. This assay can be updated or modified seasonally as new infections emerge to appropriately survey the current pathogen climate. Improvements made by this assay over current workflows will not only provide greater insight into a patient's humoral immune system, but will also help inform vaccine development. For example, a patient's antibody response or circulating antibody population can be assessed for binding to various candidate antigens.

Detector molecular assemblies 46 may all be of the same type or may be detected using the same detection method. The detected signal may not contain any unique barcode information. In one example, the detector tail may contain a reactive molecule that generates a signal. In one embodiment, detector molecular assembly 46 is detected based on enzymatic conversion of a substrate to an optically detectable product. Optical detection involves spots on an array of supramolecular structures (40). In one embodiment, the detector molecules 1 of each detector molecule assembly 46 may all be of the same type and/or have the same binding specificity. In one example, the detected analyte molecules 44 are all human antibodies and the detector molecule is an anti-human antibody with general binding specificity for a broad range of human antibodies regardless of antigen specificity. Other embodiments are also contemplated. For example, the analyte molecule 44 may undergo a tagging or processing step to add a tag (e.g., biotin) that allows binding to the streptavidin detector molecule 1. In the depicted embodiment, the steps of providing the detector molecule assembly 46 may be less complex, since the pool of detector molecule assemblies 46 is not diverse, and the detector molecule 1 and the associated tail or linker are also all This is because they may be of the same type. Additionally, the detection step may also be less complex since no sequence or barcode information is obtained from the detector side. Accordingly, in one embodiment, the unique identifying information is the capture barcode 20 used in conjunction with the detector-generated signal location and location information for each capture barcode. Analyte binding is characterized using correlation of the detector signal with the position of a specific barcode. It should be understood that the analyte detection method of FIG. 9 can also be performed using a diverse pool of detector molecular assemblies having unique barcodes as well as reactive molecules that produce a detector signal.

Figure 10 shows an embodiment similar to the analyte detection method of Figure 9, but with varying resolution of the detector molecular assembly 46. The various pools of detector molecule assemblies 46 carry different respective detector molecules 1 and detector barcodes 21 (shown as detector barcodes 21a, 21b, 21c, and 21d). The combined detector structure 50 includes a unique capture barcode 20 associated with a capture molecule 2 specific for a particular analyte molecule 44 and a unique detector barcode 21 specific for the analyte molecule 44. Includes both. Either the unique capture barcode 20 or the unique detector barcode 21 or both can be evaluated to characterize analyte binding. Detection of unique detector barcode 21 may be performed using techniques discussed with respect to unique capture barcode 20, including optical, electrical and/or magnetic detection. Detection may include generating sequence data or amplification data of a unique detector barcode 21.

In some embodiments, a sample comprising one or more analytes is contacted with one or more supramolecular structures 40. In some embodiments, as described herein, a plurality of supramolecular structures are provided attached to one or more solid substrates. Figures 11-14 provide examples of supramolecular structures attached to patterned solid substrates. Figure 15 shows an example of a supramolecular structure incorporated into a porous hydrogel matrix. The disclosed techniques can be performed with patterned substrates comprising binding sites distributed on or within the binding sites. In one embodiment, each binding site accommodates a single supramolecular structure 40 with differential chemistry. Patterned substrates can be fabricated through lithographic processes. Additionally, embodiments of the disclosed technology may include one or more regeneration steps to remove bound or “spent” bound detector structures 50 from the substrate for incorporating new supramolecular structures 40.

11 shows the detection of analyte molecules in a sample (i.e., at single molecule resolution) using a surface-based assay using a supramolecular structure 40 as described herein for single-molecule counting of analytes in a sample. Provides an exemplary illustration of a method for detecting analyte molecules in a sample. In some embodiments, the supramolecular structure comprises a core structure comprising a DNA origami core. In some embodiments, (a) a fiducial marker 62 that serves as a reference coordinate for all features on the substrate 60; (b) a set of defined micropatterned binding sites 66 to which individual core structures (e.g., DNA origami) can be anchored; (c) A planar substrate (60) is provided that includes a background passivation (64) that minimizes or prevents interaction between the supramolecular structures (including capture molecules, core structure molecules) and the surface of the substrate (60). In some embodiments, a fiducial marker comprises a defined geometric feature on the surface to be used as a fiducial feature for other features on the substrate. In some embodiments, fiducial marker 62 is coated with a polymer or self-assembling monolayer that does not interact with other molecules (e.g., DNA origami) or the core structure of the supramolecular structure. In some embodiments, background passivation 64 minimizes or prevents interaction between the surface of substrate 60 and analyte molecules in the sample. In some embodiments, planar substrate 60 includes an optical or electrical device, such as a FET, ring resonator, photonic crystal, or microelectrode, that is defined prior to formation of binding site 66. In some embodiments, binding sites 66 are micropatterned on a planar substrate 60. In some embodiments, the binding sites 66 on the surface are in a periodic pattern. In some embodiments, the binding sites 66 on the surface are in a non-periodic pattern (e.g., random). In some embodiments, the minimum distance between any two binding sites 66 is specified. In some embodiments, the minimum distance between any two binding sites 66 is at least about 200 nm. In some embodiments, the minimum distance between any two binding sites 66 is at least about 40 nm to about 5000 nm. In some embodiments, the geometry of binding site 66 includes a circle, square, triangle, or other polygonal shape. In some embodiments, the chemical groups used in passivation 64 include neutrally charged molecules such as tri-methyl silyl (TMS), uncharged polymers such as PEG, zwitterionic polymers such as or combinations thereof. In some embodiments, the chemical groups used to define binding site 66 include silanol groups, carboxyl groups, thiols, other groups, or combinations thereof.

In some embodiments, a single supramolecular structure 40 is attached to each binding site 66 (step 1). Reference numeral 70 provides a drawing showing the components of supramolecular structure 40 individually and as assembled and arranged on a planar substrate (components as described herein, e.g., Figures 1-4). equivalence). In some embodiments, the supramolecular structure 40 includes a core structure 13 comprising a DNA origami, where the supramolecular structure 40 is attached to each binding site using a DNA origami placement technique (Step 1). . In some embodiments, supramolecular structure 40 is assembled prior to attachment to each binding site 66. In some embodiments, the DNA origami comprises a unique shape and dimension to facilitate binding to a binding site using DNA origami placement techniques. In some embodiments, DNA origami placement involves directed self-assembly techniques to organize individual DNA origami (e.g., core structures) on a surface (e.g., a micropatterned surface). In some embodiments, alternatively to the DNA origami arrangement, the reactive groups of the supramolecular nanostructure 40 are bound to a pre-organized DNA origami on the binding site. In some embodiments, both of these methods of binding supramolecular nanostructures to corresponding binding sites rely on the ability to organize one or more molecules onto micropatterned binding sites using DNA origami placement techniques. In some embodiments, the planar substrate can be stored for a considerable period of time in a clean environment after this step.

With continued reference to Figure 11, in some embodiments, a sample comprising analyte molecules (as described herein) is contacted with a planar substrate in an analyte capture step (step 2). In some embodiments, the sample is contacted with a planar substrate using a flow-cell. In some embodiments, the sample is incubated on a planar substrate with a supramolecular structure attached to the binding site 66. In some embodiments, the incubation period can be from about 30 seconds to about 24 hours. In some embodiments, the incubation period is about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 minutes. Hours to about 12 hours, about 12 hours to about 24 hours, about 24 hours to about 48 hours.

In some embodiments, analyte molecules 44 in the sample interact with supramolecular structures 40 on planar surface 60. In some embodiments, a single copy of a specific analyte molecule 44 binds to a capture molecule. Still referring to Figure 11, in step 3, the detector molecular assembly 46 is contacted with the captured analyte. The detector molecular assembly is detected in step 4 to produce a detectable signal. For example, the detector barcode 21 is used as a binding site for a signaling element 76 that contacts the associated detector molecular assembly 46. In some embodiments, signaling elements 76 include fluorescent molecules or microbeads, fluorescent polymers, highly charged nanoparticles or polymers. In some embodiments, one or more signaling elements 76 are allowed to interact with the supramolecular structure on the planar structure. In some embodiments, signaling element 76 is introduced into a flow-cell containing a planar substrate. In some embodiments, the detector barcode is amplified as set forth in the depicted embodiment. For example, detector barcodes are used as polymerization initiators for the growth of highly fluorescent polymers in processes such as rolling circle amplification or hybridization chain reaction. In some embodiments, the detectable signal as described in step 4 is a surface such that all individual analyte capture events result in the presence of a signaling element 76 at the location of each analyte (linked to a capture molecule and a detector molecule). It continues.

In some embodiments, signaling element 76 is optically active and can be measured using a microscope or optical sensor integrated within planar substrate 60. In some embodiments, the signaling element is electrically active and can be measured using an integrated electrical sensor. In some embodiments, signaling element 76 is magnetically active and can be measured using an integrated magnetic sensor. In some embodiments, each signaling event is associated with capture of the same type of analyte molecule (a single copy of the same type of analyte molecule) as determined by the corresponding detector and capture molecule, and thus signaling element 76 Counting the number of positions present provides quantification of the analyte molecules in the sample.

Figure 12 shows an arrangement with a similar planar substrate 60 having binding sites 66 as in Figure 11. The planar substrate 60 has the assembled supramolecular structures 40 secured to each binding site 66 (step 1). After analyte capture (step 2), the detector molecular assembly is brought into contact with the captured analyte molecules 44. Here, each detector molecular assembly 46 includes an associated core structure (e.g., core structure 13, see FIG. 1), such as a DNA origami, that acts as an integral signaling element 76. Figure 13 shows an arrangement with a planar substrate 60 (which may be formed as described with respect to Figure 11) having an assembled supramolecular structure 40 secured to each binding site 66 (Step 1). . A separate process associates the analyte 44 with each detector molecular assembly 46. In one embodiment, each detector molecular assembly 46 possesses a signal element 76, such as a supramolecular core structure. The associated analyte and detector molecular assembly (46) is incubated with the planar substrate (60) and the immobilized supramolecular structure (40). The associated analyte and detector molecular assembly 46 binds to a supramolecular structure with a capture molecule having binding specificity for the analyte, and the bound analyte may be, for example, a signaling element (which may be part of the detector molecular assembly 46). or can be characterized through detecting the signal generated from the analyte and the detector molecule assembly 46, which can be added after association of the capture molecule.

14, a patterned substrate 60 with multiple binding sites 66 can be functionalized with or connected to individual supramolecular structures 40. In the depicted embodiment, the supramolecular structure 40 is assembled in step 80 and then arranged on a substrate 60 before the antigen capture molecule 2 is linked. For example, a DNA origami containing specific barcodes and capture strands specific for a particular antigen is flowed over the substrate 60 and placed on a single molecule array, e.g., DNA binding features. The antigen is conjugated in step 82 to include a complementary strand presented as a capture molecule (2), which can anneal to the DNA origami barcode (20) of the supramolecular structure. Alternatively, the capture molecule is associated with the supramolecular structure 40 during assembly (step 80) and applied to the substrate 60 along with the supramolecular structure 40. The barcode 20 of each supramolecular structure is read through sequencing, amplification, or hybridization assay. A map of the antigen capture molecules 2 with their spatial positions on the substrate 60 can be obtained before performing the assay and performed as a quality control of the substrate 60. The map can be stored in an analyte detection system as provided herein (see Figure 16) and used to generate reports of positive binding events to provide diagnostic information.

Then, in step 84, appropriate blocking conditions are added and a sample comprising patient antibodies (e.g., serum) is applied to the substrate 60 and incubated to allow the antibody analytes 44 in the sample to form antibody-antigen complexes. can be formed. The sample is then washed and a detector molecule 1, a secondary anti-human antibody, and a detector molecule assembly 46 comprising a label for detection are added in step 86. Detection of the bound detector structure 50 (step 88) may be based on detection of an antibody label, which may be DNA-based for amplification via rolling circle amplification or hybrid chain reaction; Alternatively, the label can be a DNA nanoparticle or a fluorescent polymer.

In one embodiment, the assay is adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus B.1.1.7, coronavirus B.1.351, coronavirus P.1 coronavirus NL63, coronavirus OC43, human metapneumovirus, Human rhinovirus/enterovirus, influenza A, subtypes 2009H1N1, H1, H3, influenza B, parainfluenza virus types 1, 2, 3 and 4, respiratory syncytial virus, Chlamydophila pneumoniae, Mycoplasma pneumoniae Ae and Bordetella pertussis. Additionally, the assay targets vaccine targets for seasonal flu, as well as COVID vaccine targets for one or more commercial vaccines (Moderna, Pfizer, Astra Zeneca, Novavax, and Johnson). May include Johnson and Johnson. The addition of these vaccine targets allows classification of immunity to determine whether a patient has immunity from natural infection and/or acquired immunity from a vaccine. Multiple antigens may be present for each pathogen to define the specificity of immunity.

Detection may include anti-species for specific antibodies for determination of IgG, IgM and/or IgA. Specific detection of subtypes helps to further understand immune maturation.

15 provides an exemplary embodiment for forming a hydrogel matrix 100, wherein in addition to combining one or more monomers 122 and one or more cross-linking molecules 124 to form a hydrogel, one or more supramolecules Structure 40 is introduced. In some embodiments, one or more supramolecular structures 40 copolymerize with a hydrogel matrix to form matrix 120. In some embodiments, each anchor molecule 18 of one or more supramolecular structures 40 copolymerizes with the hydrogel matrix 120. In some embodiments, one or more monomers 122 include acrylamide. In some embodiments, the one or more crosslinking agents include bis-acrylamide.

Embodiments of the present disclosure include one or more computer-implemented detection systems configured to perform certain methods of the disclosed embodiments. 16 shows an analyte detection system 1000 including a controller 1001. Controller 1001 includes a processor 1002 and a memory 1004 that stores instructions configured to be executed by processor 1002. Controller 1001 includes a user interface 1006 and communication circuitry to facilitate communication, for example, over the Internet 1010 and/or over a wireless or wired network. User interface 1006 facilitates user interaction with characterized analyte detection results as provided herein.

Processor 1002 is programmed to receive analyte detection data and characterize the detected analytes. In one embodiment, the processor generates a report of detected analytes in the sample following incubation of the array of supramolecular structures and detection of the detector molecular assembly. The report may include data of the generated optical signals at various binding sites corresponding to the detected analyte binding events. The report may include processed data, such as a list of detected analytes or combined positive/negative results. The report may include a list of available capture molecules on the array indicating the analyte detection capability.

System 1000 also includes an analyte detector 1020 operative to detect analyte binding through detection of one or more components of the supramolecular structure. Analyte detector 1020 includes a detection system having one or more sensors 1022. Analyte detector 1020 may also include a reaction controller 1024 that controls sample incubation and appropriate release of reaction reagents and detector molecular assemblies at appropriate times. Sensor 1022 may be one or more of an optical sensor (eg, a fluorescence sensor, an infrared sensor), an image sensor, an electrical sensor, or a magnetic sensor. In one embodiment, sensor 102 is a metal-oxide semiconductor image sensor device.

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

Claims (42)

A method for detecting analyte molecules present in a sample, comprising:
A step of providing an array of supramolecular structures fixed on a substrate or in a porous matrix, wherein the supramolecular structures of the array are
A core structure comprising a plurality of core molecules; and
Capture molecule linked to core structure via capture barcode
A step comprising;
contacting the sample with the array such that the analyte molecules bind to the capture molecules;
contacting the analyte molecule bound to the capture molecule with the detector molecule assembly such that the detector molecule of the individual detector molecule assembly binds to the analyte molecule to form a bound detection structure;
detecting analyte molecules based on signals generated by individual detector molecule assemblies of the coupled detection structure; and
Step of associating detected analyte molecules and supramolecular structures based on the identity of the capture barcode
How to include . The method of claim 1 , further comprising, after contacting the sample with the array, removing additional analyte molecules in the sample that are not bound to the supramolecular structures of the array. The method of claim 1 , further comprising removing detector molecular assemblies that are not bound to the supramolecular structures of the array after forming the bound detection structures. The method of claim 1 , wherein the analyte molecule comprises a protein, peptide, peptide fragment, lipid, DNA, RNA, organic molecule, inorganic molecule, complex thereof, or any combination thereof. The method of claim 1, wherein the supramolecular structure of the array is a nanostructure. 6. The method of claim 5, wherein the core structure is a nanostructure. The method according to claim 1, wherein the plurality of core molecules of the core structure are arranged in a predefined shape and/or have a defined molecular weight. 8. The method of claim 7, wherein the predefined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. The method of claim 1, wherein the core structure is a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tile structure, or a multi-stranded structure. A method comprising a DNA tile structure, a single-stranded RNA origami, a multi-stranded RNA tile structure, a hierarchically organized DNA or RNA origami with multiple scaffolds, a peptide structure, or a combination thereof. 2. The method of claim 1, wherein the capture barcode comprises a first capture linker, a second capture linker, and a capture bridge disposed between the first capture linker and the second capture linker, wherein the first capture linker is a first capture linker on the core structure. A method wherein the capture molecule and the second capture linker are linked together via a bond to a third capture linker. The method of claim 1, wherein the capture molecule of the individual supramolecular structure has binding specificity for the analyte molecule, and the second capture molecule of at least one other supramolecular structure of the array does not have binding specificity for the analyte molecule. method. The method of claim 1 , wherein each capture molecule of the array of supramolecular structures has a different binding specificity for each different analyte molecule. The method of claim 1 , wherein a detector molecule in the assembly of detector molecules has binding specificity for an analyte molecule and the other detector molecules in the assembly of detector molecules do not have binding specificity for the analyte molecule. The method of claim 1 , wherein the detector molecules of the detector molecule assembly have different binding specificities for each different analyte molecule. The method of claim 1, wherein the detector molecule of the detector molecule assembly has binding specificity for any analyte molecule bound to the capture molecule of the supramolecular structure. The method of claim 1, wherein the detector molecules of the detector molecule assembly do not have binding specificity for the supramolecular structure. The method of claim 1, comprising determining the identity of the supramolecular structure by determining the nucleic acid sequence of the capture barcode. 18. The method of claim 17, comprising correlating the determined nucleic acid sequence of the supramolecular structure to a position on the array. 19. The method of claim 18, wherein associating the detected analyte molecule with the supramolecular structure based on the identity of the capture barcode comprises associating the location of the signal with a location on an array of nucleic acid sequences. 19. The method of claim 18, wherein the signal is an optically, magnetically and/or electrically detectable signal generated by a reactive molecule of the detector molecular assembly. 19. The method of claim 18, wherein the signal is a sequence of a detector barcode of a detector molecular assembly. An array for detecting one or more analyte molecules in a sample, comprising:
write;
Multiple supramolecular structures fixed on a substrate
It includes, where the individual supramolecular structures of the plurality of supramolecular structures are
(a) a core structure comprising a plurality of core molecules, coupled to a substrate or connected to the substrate by anchor molecules,
(b) a capture barcode coupled directly or indirectly to the core structure at a first end of the capture barcode, the capture barcode generally extending away from the substrate; and
(c) a capture molecule coupled to the capture barcode at a second end of the capture barcode, wherein the capture molecule is configured to bind to an analyte molecule.
which includes
Array. The substrate according to claim 22, wherein each core structure of the plurality of supramolecular structures is identical to each other. 23. The substrate of claim 22, wherein each supramolecular structure has a unique capture barcode. 23. The substrate of claim 22, comprising a solid support or a porous matrix. The substrate according to claim 22, wherein each supramolecular structure is a nanostructure. 23. The substrate of claim 22, wherein each core structure is a nanostructure. 23. The substrate of claim 22, wherein the plurality of core molecules for each core structure are arranged in a predefined shape and/or have a defined molecular weight. 23. The substrate of claim 22, wherein the core structure is directly coupled to the substrate. The substrate of claim 22, comprising a plurality of analyte molecules bound to each of the plurality of supramolecular structures. 31. The substrate of claim 30, comprising a plurality of detector molecule assemblies coupled to each analyte molecule of the plurality of analyte molecules. 32. The substrate of claim 31, wherein each detector molecule assembly comprises a detector molecule coupled to a detector barcode comprising one or more linkers. 34. The substrate of claim 33, wherein each detector molecule assembly comprises a core structure coupled to the detector molecule by a detector barcode. 23. The substrate of claim 22, comprising a porous matrix. 35. The substrate of claim 34, wherein the porous matrix comprises a hydrogel. 23. The substrate of claim 22, comprising a planar substrate. 23. The substrate of claim 22, wherein the individual supramolecular structures comprise only one capture molecule. As a substrate for detecting one or more analyte molecules in a sample,
A patterned substrate comprising a plurality of binding sites spaced apart from each other; and
A single supramolecular structure associated with each binding site of a plurality of binding sites.
It includes, and the supramolecular structure is
A core structure comprising a plurality of core molecules, coupled to a substrate or connected to the substrate by an anchor molecule,
a capture barcode coupled directly or indirectly to the core structure at a first end of the capture barcode, the capture barcode generally extending away from the substrate; and
A capture molecule coupled to the capture barcode at a second end of the capture barcode, wherein the capture molecule is configured to bind to an analyte molecule.
which includes
write. 39. The method of claim 38, comprising one or more fiducial markers disposed on or within the patterned substrate, wherein the one or more fiducial markers are detectable by a detection system to provide positional information for the supramolecular structure. phosphorus description. 39. The substrate of claim 38, comprising a passivated region on the patterned substrate separating a plurality of binding sites. The substrate of claim 38, wherein each binding site of the plurality of binding sites has a size to accommodate a single supramolecular structure. An array for detecting one or more analyte molecules in a sample, comprising:
A patterned substrate comprising a plurality of binding sites; and
Supramolecular structure associated with each binding site of a plurality of binding sites
It includes, and the supramolecular structure is
A core structure comprising a plurality of core molecules, coupled to a substrate or connected to the substrate by an anchor molecule,
a capture barcode coupled directly or indirectly to the core structure at a first end of the capture barcode, the capture barcode generally extending away from the substrate; and
A capture molecule coupled to the capture barcode at a second end of the capture barcode, wherein the capture molecule is configured to bind to an analyte molecule.
which includes
Array.

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