SOLUTION PHASE SINGLE MOLECULE CAPTURE AND ASSOCIATED TECHNIQUES
Provided herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, the one or more analyte molecules form a complex in solution with a supramolecular structure. The supramolecular structures of the complex may be detectable such that binding of the analyte molecule to a binding site of an array is detectable via one or more features of the supramolecular structure. A binding site of an array includes capture molecules to capture bound complexes to facilitate detection.
1 . A method for detecting an analyte molecule present in a sample, the method comprising:
providing a sample comprising analyte molecules;
contacting the sample with a pool of supramolecular structures in solution to form analyte molecule-supramolecular structure complexes, an individual analyte molecule-supramolecular structure complex comprising:
a core structure comprising a plurality of core molecules;
an affinity binder linked to the core structure; and
an analyte molecule of the analyte molecules bound to the affinity binder,
wherein different supramolecular structures of the pool of supramolecular structures comprise different affinity binders with different binding affinity for other analyte molecules of the analyte molecules;
contacting the analyte molecule-supramolecular structure complexes with an array,
wherein binding sites of the array comprise respective immobilized affinity binders with binding affinity for different analyte molecules; and
detecting binding of the analyte molecule-supramolecular structure complexes to binding sites of the array.
2 . The method of claim 1 , comprising identifying the individual analyte molecule of the individual analyte molecule-supramolecular structure bound to the individual binding site of the array.
3 . The method of claim 2 , wherein the identifying comprises: generating a detectable signal from the supramolecular structure and associating the detectable signal with the individual binding site.
4 . The method of claim 3 , wherein the detectable signal is an optical, magnetic, or electrical signal indicative of a presence of the individual analyte molecule-supramolecular structure at the individual binding site.
5 . The method of claim 2 , wherein the identifying comprises: amplifying an initiator of the supramolecular structure and detecting the amplifying.
6 . The method of claim 5 , wherein the initiator is a nucleic acid.
7 . The method of claim 2 , wherein the identifying comprises: amplifying an initiator immobilized on the individual binding site and detecting the amplifying.
8 . The method of claim 7 , wherein the initiator is a nucleic acid.
9 . The method of claim 1 , wherein the affinity binder and the immobilized affinity binders are antibody molecules or portions of antibody molecules.
10 . The method of claim 1 , wherein binding of the analyte molecule-supramolecular structure to an immobilized affinity binders at an individual binding site comprising binding of the affinity binding of the supramolecular structure to a first portion of the analyte molecule and binding of the immobilized affinity binder to second portion of the analyte molecule.
11 . The method of claim 1 , wherein the immobilized affinity binders are linked to each individual binding site via nucleic acid capture molecules.
12 . The method of claim 11 , wherein each individual binding site comprises a plurality of nucleic acid capture molecules.
13 . The method of claim 12 , wherein the plurality of nucleic acid capture molecules at an individual binding site all have a same nucleic acid sequence.
14 . The method of claim 12 , wherein the plurality of nucleic acid capture molecules are coupled to an immobilized supramolecular structure.
15 . The method of claim 1 , wherein each individual binding site comprises a plurality of immobilized affinity binders all having affinity for a same analyte molecule of the analyte molecules.
16 . The method of claim 1 , wherein each binding site has a diameter between 20-500 nanometers.
17 . The method of claim 1 , wherein each supramolecular structure of the pool is a nanostructure.
18 . The method of claim 14 , wherein each core structure is a nanostructure.
19 . The method of claim 1 , wherein each supramolecular structure of the pool is arranged into a pre-defined shape and/or have a prescribed molecular weight.
20 . The method of claim 1 , further comprising removing analyte molecule-supramolecular structures not bound to binding sites of the array after contacting the sample with the array.
21 .- 36 (canceled)
37 . An array for analyte detection, comprising:
a plurality of binding sites patterned on a substrate and separated by interstitial surfaces of the substrate, wherein each binding site comprises a well, and wherein at least one surface of the well comprises a first chemical group and the interstitial surfaces comprise a second chemical group;
a bead loaded into each of the binding sites, wherein the first chemical group selectively binds to the bead and the second chemical group does not interact with or bind to the bead, and wherein each bead comprises:
a plurality of single-stranded oligonucleotides immobilized on each bead such that an individual binding site of the array comprises a plurality of single-stranded oligonucleotides comprising a same sequence that is distinguishable from sequences of other single-stranded oligonucleotides immobilized on different binding sites of the array;
affinity binders captured at binding sites of the array such that the captured affinity binders form capture molecules immobilized at respective binding sites; and
analyte molecules bound to individual capture molecules at the individual binding site, wherein each analyte molecule is complexed with a supramolecular structure detection assembly.
38 . A method for detecting an analyte molecule present in a sample, the method comprising:
providing a plurality of supramolecular structures, wherein each supramolecular structure comprises
a core structure comprising a plurality of core molecules;
an antibody having binding affinity for an antigen and coupled to a nucleic acid capture strand; and
a complementary strand to the nucleic acid capture strand, wherein the complementary strand is linked to the core structure and forms a duplex structure with the nucleic acid capture strand to couple the antibody to the core structure, wherein the plurality of supramolecular structures comprise different antibodies relative to one another with respective different binding affinities;
contacting the plurality of supramolecular structures with a sample comprising the antigen such that the antigen binds to the antibody to form a complex;
contacting the complex with a bead carrying a capture antibody having binding specificity for the antigen to form a sandwich structure;
displacing the nucleic acid capture strand from the complementary strand using a displacing strand to release the core structure into solution, wherein the released core structure is not coupled to the antibody; and
detecting the core structure.
39 . The method of claim 38 , wherein the core structure is a box origami formed from nucleic acids.
40 . The method of claim 39 , wherein the box origami is coupled to a plurality of fluorophores that fluoresce at a particular wavelength, and wherein detecting the core structure comprises detecting fluorescence at the particular wavelength.
41 . The method of claim 38 , wherein the capture strand comprises a toehold region that does not bind to the complementary strand but that does bind to the displacing strand.
42 . The method of claim 38 , wherein the complementary strand comprises a toehold region that does not bind to the capture strand but that does bind to the displacing strand.
43 . The method of claim 38 , comprising separating a remaining portion of the sandwich structure from the released core structure before the detecting.
44 . (canceled)