WO2023049355A1

WO2023049355A1 - Monofunctional particles

Info

Publication number: WO2023049355A1
Application number: PCT/US2022/044548
Authority: WO
Inventors: Ashwin Gopinath
Original assignee: Palamedrix, Inc.
Priority date: 2021-09-23
Filing date: 2022-09-23
Publication date: 2023-03-30

Abstract

Provided herein in various embodiments, is a method for detecting and/or quantifying an analyte molecule present in a sample without employing a sequencing operation. As discussed, monofunctional particles are used to perform the detection and/or quantification of the analyte of interest. In one embodiment the monofunctional particles include a supramolecular structure (e.g., a nucleic acid origami structure) that comprises a core structure composed of one or more core molecules, a single affinity binder linked to the supramolecular structure at a first location, and one or more unique identifiers also attached to the supramolecular structure and which convey information about the affinity binder present on a respective monofunctional particle.

Description

MONOFUNCTIONAL PARTICLES

BACKGROUND

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/247,580, entitled “MONOFUNCTIONAL PARTICLES”, filed September 23, 2021, which is herein incorporated by reference in its entirety for all purposes.

[0002] The current state of personalized healthcare is generally genome-centric, focused on quantifying the genes present within an individual. While such an approach has proven to be extremely powerful, it does not provide a clinician with the complete picture of an individual’s health. This is because, while genes are the “blueprints” of an individual, they merely inform the likelihood of developing an ailment. Within an individual these “blueprints” first need to be transcribed into RNA and then translated into various protein molecules in order to have an effect on the health of an individual.

[0003] The concentration of proteins, the interaction between the proteins (proteinprotein interactions or PPI), as well as the interaction between proteins and other molecules, are intricately linked to the health of different organs, homeostatic regulatory mechanism as well as the interaction of these systems with the external environment. Hence, identification of one or more proteins present within a patient sample (e.g., a blood sample or biopsy), as well as quantification (either absolute or relative) of such proteins, is useful to create a complete picture of an individual’s health at a given time as well as to predict any emerging health issues. For instance, the amount of stress experienced by cardiac muscles (e.g. during a heart attack) can be inferred by measuring the concentration of troponin I/II and myosin light chain present within peripheral blood. Similar protein biomarkers have also been identified, validated and are deployed for a wide variety of organ dysfunctions (e.g. liver disease and thyroid disorders), specific cancers (e.g. colorectal or prostate cancer), and infectious diseases (e.g. HIV and Zika). The interactions between these proteins are also useful for drug development and are a valuable dataset. The ability to detect and quantify proteins and protein interaction with other molecules within a given sample of bodily fluids is an integral component of such healthcare development.

BRIEF DESCRIPTION OF THE DRAWINGS

[0004] 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.

[0005] FIG. 1 depicts a process flow illustrating steps in the generation of a monofunctional particle, in accordance with aspects of the present disclosure;

[0006] FIG. 2 visually illustrates aspects of the creation of a monofunctional particle, in accordance with aspects of the present disclosure;

[0007] FIG. 3 visually illustrates aspects of attaching fluorophores to a monofunctional particle, in accordance with aspects of the present disclosure;

[0008] FIG. 4 depicts a an additional implementation of a process flow illustrating steps in the generation of a monofunctional particle, in accordance with aspects of the present disclosure;

[0009] FIG. 5 visually illustrates an example of a rolling circle amplification template, in accordance with aspects of the present disclosure;

[00010] FIG. 6 visually illustrates aspects of the creation of a monofunctional particle in accordance with the steps of FIG. 4, in accordance with aspects of the present disclosure;

[00011] FIG. 7 visually illustrates an expanded view of a strand associated with a nanoball formed using the rolling circle amplification template of FIG. 5, both before and after association of complementary fluorophores, in accordance with aspects of the present disclosure;

[00012] FIG. 8 depicts a process flow illustrating steps in the detection and/or quantification of analytes of interest using flow cytometry and monofunctional particles, in accordance with aspects of the present disclosure; [00013] FIG. 9 visually illustrates aspects of the detection and/or quantification process of FIG. 8, in accordance with aspects of the present disclosure;

[00014] FIG. 10 depicts a process flow illustrating steps in the detection and/or quantification of analytes of interest using area-based imaging and monofunctional particles, in accordance with aspects of the present disclosure;

[00015] FIG. 11 visually illustrates aspects of the detection and/or quantification process of FIG. 10, in accordance with aspects of the present disclosure; and

[00016] FIG. 12 shows a block diagram of an example processing system according to embodiments of the present disclosure.

SUMMARY

[00017] The present disclosure generally relates to systems, structures and methods for detection and quantification of analyte of interest (e.g., analyte molecules) in a sample.

[00018] Provided herein in various embodiments, is a method for detecting and/or quantifying an analyte molecule present in a sample without employing a sequencing operation. As discussed, monofunctional particles are used to perform the detection and/or quantification of the analyte of interest. In one embodiment, and as described in greater detail below, the monofunctional particles include a supramolecular structure (e.g., a nucleic acid origami structure) that comprises a core structure composed of one or more core molecules. The monofunctional particles each also comprise a single affinity binder (which may also be characterized as a reactive group herein) linked to the supramolecular structure at a first location and a one or more different types (e.g., three to eight) of unique identifiers also attached to the supramolecular structure and which convey information about the affinity binder present on a respective monofunctional particle. [00019] By way of example, the unique identifiers may, alone or taken in the aggregate, function as “barcode(s)” and may incorporate different fluorescent molecules that emit, when excited, at a known frequency or frequency range and/or that emit only when excited by radiation (e.g., light) at a known frequency or frequency range). As used herein a “barcode” may be understood to be a molecule or structure that either alone or in combination with other barcodes provides unique identifying information that may be used to identify or characterize a monofunctional particle 100 as having a particular affinity binder attached, i.e., as being specific to a respective analyte molecule. By way of example, in a single unique identifier context a known frequency of emission associated with a unique identifier may correspond to a known affinity binder. In other contexts, different combinations of emitted frequencies and/or proportions of emitted frequencies may be associated with respective affinity binders.

[00020] The monofunctional particles may be contacted with a sample that potentially contains the analyte molecule (e.g., protein) of interest, such as in a solution phase. In certain embodiments, an excitation/emission based detection scheme may then be employed (such as flow cytometry) to detect and/or quantify instances of analyte binding events to the monofunctional particles based on the excitation and emissions of the unique identifiers. It should be appreciated, however, that the preceding explanation and example are provided by way of non-limiting illustration only. In practice, the present approaches cover any method used to classify, identify, and/or quantify analyte molecules of interest using monofunctional particles as discussed herein.

[00021] For example, instead of multiple copies of different types of unique identifiers (e.g., barcodes) being attached to the supramolecular structure of the monofunctional particle, the unique identifiers may instead be incorporated into a circular template molecule (e.g., a rolling circle amplification template) that is attached to the supramolecular structure at a location separate from the affinity binder linkage. Incubation of the circular template molecule with suitable primers yields a nanoball attached to the supramolecular structure that, when functionalized, fluoresces at frequencies determined by the sequences provided on the circular template molecule. Unlike the prior example, use of a nanoball signal molecule allows emissive optical signal strength to be adjusted upwards or downwards by adjusting incubation period and/or conditions. In contrast, embodiments employing fixed numbers of copies of the different types of unique identifiers are fixed or limited in terms of the absolute value of optical signal produced.

[00022] As discussed herein, monofunctional particles may be used in various techniques for detecting an analyte molecule of interest when the analyte molecule is present in the sample at a count of a single molecule or higher. Further, with respect to examples and explanations as discussed herein, a respective sample may comprise a complex biological sample and methodologies utilizing a monofunctional particle may increase the dynamic range of a detection operation and/or facilitate quantitative assessment of a range of molecular concentrations within the complex biological sample.

[00023] By way of example, as used herein a sample may comprise a biological sample, such as an aqueous solution comprising a protein, a peptide, a fragment of a peptide, a lipid, DNA, RNA, an organic molecule, a viral particle, an exosome, an organelle, or any complexes thereof. In some embodiments, the sample comprises or is derived from a tissue biopsy, blood, blood plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultures cells, culture media, discarded tissue, plant matter, synthetic proteins, prions, a bacterial and/or viral sample or fungal tissue, or combinations thereof. The sample may be isolated from a primary source such as cells, tissue, bodily fluids (e.g., blood), environmental samples, or combinations thereof, with or without purification. In embodiments where cells are involved in sample preparation the cells may be lysed using a mechanical process or other cell lysis methods (e.g., lysis buffer). The sample may be filtered using a mechanical process (e.g., centrifugation), micron filtration, chromatography columns, other filtration methods, or combinations thereof. Further, the sample may or may not be treated with one or more enzymes to remove one or more nucleic acids or one or more proteins. In certain implementations the sample is collected from one or more individual persons, one or more animals, one or more plants, or combinations thereof. By way of example, the sample may be collected from an individual person (e.g., a patient of subject), animal and/or plant having a disease or disorder that comprises an infectious disease, an immune disorder, a cancer, a genetic disease, a degenerative disease, a lifestyle disease, an injury, a rare disease, an age-related disease, or combinations thereof.

[00024] The sample may further be an environmental sample, such as a wastewater or soil sample. Further, the sample may also be a non-biological sample. In an embodiment, the sample may be a sample from a chemical process step, a sample of food or nutritional components, or packaging components. In some embodiments, the analyte molecule of interest within a given sample may comprise a protein, a peptide, a peptide fragment, a lipid, a DNA, a RNA, an organic molecule, an inorganic molecule, complexes thereof, or any combinations thereof.

[00025] In some embodiments, each core structure of a respective supramolecular structure forming a monofunctional particle is a nanostructure. In certain implementations, each core structure of a plurality of supramolecular structures (e.g., a plurality of monofunctional particles) are identical to each other. In some embodiments, each supramolecular structure of the monofunctional particles comprises a prescribed shape, size, molecular weight, or combinations thereof, so as to reduce or eliminate crossreactions between a plurality of supramolecular structures. In some embodiments, the one or more 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. By way of example, each core structure may independently comprises a deoxyribonucleic acid (DNA) origami (e.g., a scaffolded DNA origami), a ribonucleic acid (RNA) origami (e.g., a scaffolded RNA origami), a hybrid DNA:RNA origami (e.g., a scaffolded hybrid (DNA:RNA) origami), a singlestranded DNA tile structure, a multi -stranded DNA tile structure, a single-stranded RNA origami, a multi-stranded RNA tile structure, hierarchically composed DNA or RNA origami with multiple scaffolds, a peptide structure, or combinations thereof.

[00026] In certain implementations the respective analyte molecule is bound to the affinity binder or reactive group of a monofunctional particle through a chemical bond. In some embodiments, the affinity binder or reactive group comprises a protein, a peptide, an antibody, an aptamer (RNA and DNA), a darpin, a polymer like PEG, or combinations thereof. In some embodiments, for each monofunctional particle the affinity binder or reactive group is linked to the core structure of the supramol ecul ar structure via complementary binding. By way of example, an affinity binder linker structure of the respective affinity binder may form a bond with a complementary linker structure bound or otherwise attached to the supramol ecul ar structure.

[00027] In some embodiments, each supramolecular structure may further comprise an anchor molecule linked to the core structure. In some embodiments, the anchor molecule comprises an amine, a thiol, a DBCO, a maleimide, biotin, an azide, an acrydite, a NHS- ester, a single stranded nucleic acid (RNA or DNA) of specific sequence, or combinations thereof. In such embodiments, the anchor molecule may facilitate binding of the monofunctional particle to respective binding sites on a surface of a substrate, such as a shaped or planar substrate. Such substrates may comprise a plurality of binding sites, wherein each binding site is configured to link with a corresponding supramolecular structure via the respective anchor molecule. When bound to the surface of the substrate the monofunctional particles may undergo a readout process, such as a series of excitation and emission steps whereby monofunctional particles attached to the substrate are interrogated to classify and/or quantify an analyte molecule. Alternatively, as described herein the monofunctional particles bound to analytes may be read-out or otherwise detected and quantified while in solution (i.e., not bound to a substrate), such as using flow cytometry.

DETAILED DESCRIPTION

[00028] Disclosed herein are structures and methods for detecting one or more types of analyte molecules present in a sample. In some embodiments, the analyte molecules are detected subsequent to capture (e.g., binding) of the analyte molecules by monofunctional particles co-mingled with the sample, such as in a solution phase. As used herein, each monofunctional particle includes as part of its structure a single affinity binder that is specific to a respective analyte molecule. Each monofunctional particles also includes one or more unique identifiers (e.g., “barcodes”) such that the barcode or combination of barcodes (e.g., the respective barcodes that are present and/or the ratio of respective barcodes that are present) is unique to monofunctional particles based on their respective analyte affinity (i.e., the attached affinity binder). The barcodes may then be read-out by a detection apparatus to classify and/or quantify analyte molecules of interest present in the sample.

[00029] Monofunctional particles as discussed herein may include a supramol ecul ar structure having a core structure composed of one or more core molecules. By way of example, the core structure may be a nucleic acid origami structure, such as a DNA origami structure. As discussed herein, a single affinity binder and one or more unique identifier(s) are attached (e.g., linked) to the supramolecular structure to form the monofunctional particle.

[00030] As discussed herein, monofunctional particles to which an analyte molecule has been bound may be detected by a suitable read-out or detection mechanism. In general, any suitable detection mechanism may be employed, including detection mechanisms based on optical, electrical, or magnetic detection schemes. For example, the monofunctional particle may remain free in a solution phase both during and after exposure to a sample, with read-out and detection being performed while the monofunctional particle is free in the solution phase, such as via flow cytometry. Alternatively, in certain embodiments the supramolecular structure of each monofunctional particle is a nucleic acid origami that may be linked to or immobilized on a substrate, before or after sample exposure, with readout occurring while the monofunctional particles are immobilized on the substrate.

[00031] Thus, as provided herein, detectable analyte binding can be associated with an individual monofunctional particle to generate detection and/or quantification results. Within a given detection and/or quantification operation a sample may be processed using a variety of sets of monofunctional particles, each set having a different affinity binder, such that each set of monofunctional particles has an affinity for a different analyte molecule of interest. Such an approach allows a sample having an uncharacterized composition of multiple possible analytes of interest to analyzed and characterized for the presence and/or concentration of particular analytes of interest. For example, a human sample can be characterized to determine a presence and/or concentration of one or more proteins, peptides, peptide fragments, lipids, nucleic acids, organic molecules, inorganic molecules, and so forth, of interest. Alternatively, as may be appreciated, if only a single analyte molecule is of interest, monofunctional particles having respective affinity binders specific to the analyte molecule of interest, may be used to process the sample.

[00032] With the preceding in mind, and turning to FIGS. 1 and 2, a process flow (FIG. 1) illustrating steps in the fabrication of one embodiment of a monofunctional particle 100 is provided along with a corresponding visual representation (FIG. 2). Monofunctional particles as discussed herein include supramolecular structures 10 on which other relevant molecular structures are attached. Turning to FIG. 1, such supramolecular structures 10 are acquired or synthesized (step 12) as part of an overall process for manufacturing monofunctional particles 100. In some embodiments, the supramolecular structure 10 is a programmable structure that can spatially organize molecules. Further, in certain implementations the supramolecular structure 10 comprises a plurality of molecules linked together, some or all of which may interact with one another.

[00033] The supramolecular structure 10 may have a specific shape or geometry, e.g., a substantially planar shape that has a longest dimension in an x-y plane. In some embodiments, the supramolecular structure 10 is a nanostructure, such as a nanostructure that comprises a prescribed molecular weight based on the plurality of molecules forming the supramolecular structure 10. The plurality of molecules may, for example, be linked together through a bond, a chemical bond, a physical attachment, or combinations thereof. In certain implementations the supramolecular structure 10 comprises a large molecular entity, of specific shape and molecular weight, formed from a well-defined number of smaller molecules interacting specifically with each other. The structural, chemical, and physical properties of the supramolecular structure 10 may be explicitly designed. By way of example, the supramolecular structure 10 may comprise a plurality of subcomponents that are spaced apart according to a prescribed distance. In some embodiments, at least a portion of the supramolecular structure 10 (or its constituent core structure) is rigid or semi-rigid. Correspondingly or alternatively, all or parts of the supram olecular structure 10 (or its constituent core structure) may be flexible or conformable. In certain embodiments the supramolecular structure 10 is at least 50 nm - 200 nm in at least one dimension. In certain embodiments the supramolecular structure 10 is at least 20 nm long in any dimension.

[00034] In general, the supramolecular structure 10 may comprise a core structure which may be a polynucleotide structure, a protein structure, a polymer structure, or a combination thereof. In some embodiments, the core structure comprises either one core molecule or two or more core molecules linked together. By way of example, the one or more core molecules may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 or 500 unique molecules that are linked together. In some embodiments, the one or more core molecules comprises from about 2 unique molecules to about 1,000 unique molecules. In certain implementations, the one or more core molecules interact with each other and define the specific shape of the supramolecular structure 10. By way of example, the plurality of core molecules may interact with each other through reversible non-covalent interactions.

[00035] In some embodiments, the specific shape of the core structure of the supramolecular structure 10 has a three-dimensional (3D) configuration. Further, the one or more core molecules may provide a specific molecular weight. For example, all core structures of a plurality of supramolecular structures 10 may have a same configuration, size, and/or weight, but may differ in their attached linker sequences and/or other attached molecules, as described herein. However, excluding such differing linkers or other attached molecules, the supramolecular structures 10 of such a plurality may be otherwise identical. In certain examples the core structure may be a nanostructure. In some cases, the one or more core molecules comprise one or more nucleic acid strands (e.g., DNA, RNA, unnatural 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 an entirely polynucleotide structure. [00036] In some embodiments, the supram olecular structure 10 or its constituent core structure(s)) comprise a deoxyribonucleic acid (DNA) origami, a ribonucleic acid (RNA) origami, a hybrid DNA/RNA origami, a single-stranded DNA origami, a single-stranded RNA origami, a hierarchically composed DNA and/or RNA origami with multiple scaffolds, a peptide structure, an enzymatically synthesized nucleic acid structure (e.g., nanoball(s)), or combinations thereof. As discussed herein, in such embodiments the DNA origami, RNA origami, or hybrid DNA/RNA origami may be scaffolded. As used herein, the term “scaffold” or “scaffolded” refers to the use or inclusion of a circular ssDNA molecule, called a “scaffold” strand, that is folded into a predefined 2D or 3D shape by interacting with two or more short ssDNA, called “staple” strands, which interact with specific sub-sections of the ssDNA “scaffold” strand. In some embodiments, the core structure comprising a DNA origami, RNA origami, or hybrid DNA/RNA origami has a prescribed two-dimensional (2D) or 3D shape.

[00037] In an example embodiment, the cores structure(s) of a supramolecular structure 10 may be a nucleic acid origami that has at least one lateral dimension between about 20 nm to about 1 pm. In an embodiment, the nucleic acid origami has at least one lateral dimension between about 20 nm to about 200 nm, about 20 nm to about 400 nm, about 20 nm to about 600 nm, about 20 nm to about 800 nm, about 100 nm to about 200 nm, 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 by way of example. Further, in certain embodiments the nucleic acid origami has at least a first lateral dimension between about 20 nm to about 1 pm and a second lateral dimension, orthogonal to the first, between about 20 nm to about 1 pm. In one implementation the nucleic acid origami has a planar footprint having an area of about 200 nm² to about 1 pm².

[00038] In some embodiments, each component (e.g., constituent component) of the supramolecular structure 10 may be independently modified or tuned. By way of example, modifying one or more of the components of the supramolecular structure 10 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 10 may modify the 2D and 3D geometry of the core structure of the supramolecular structure 10. In some embodiments, such capability for independently modifying the components of the supramolecular nanostructure enables precise control over the organization of one or more supramolecular structures 10.

[00039] With the preceding high-level discussion of supramolecular structures 10, as used herein, in mind, an example of a practical implementation of a synthesis step 12 of a suitable supramolecular structure 10 is provided. In this example, the synthesized supramolecular structure 10 is a scaffolded DNA origami. In such an example a scaffold (e.g., a circular ssDNA molecule of known sequence, which may be referred to as a “scaffold” strand) may be combined with a plurality of “staples” (e.g., two or more short ssDNA, called “staple” strands, which interact with specific sub-sections of the ssDNA “scaffold” strand). The staples selectively bind to specified locations on the scaffold such that a self-assembly of the supramolecular structure 10, e.g., a DNA origami in this example, is performed. In particular, the self-assembly step results in the scaffold being folded into a predefined 2D or 3D shape via interactions with the staples. In one embodiment, the staples are formed with excess thymine (T) located at nicks as well as crossovers so as to facilitate the crosslinking (i.e., formation of covalent crosslinks) of the staple structures forming the DNA origami when exposed to an energy source (e.g., UV illumination). Such cross-linking may help improve the thermostability of the formed DNA origami. In practice, the cross-linking step may be performed after the DNA origami is purified away from any unattached staple strands.

[00040] As noted above, the monofunctional nature of the particle 100 indicates that each supramolecular structure 10 binds to a single analyte molecule of interest (or a specific region or domain of such an analyte) due to a single affinity binder being linked to the supramolecular structure 10. Such a single analyte molecule binding event might typically be associated with a relatively small or low signal strength. In order that the monofunctional particle 100 be a strong signal source, the supramolecular structure 10, in the embodiment illustrated in FIG. 1, is bound to one or more types (e.g., 3 to 8) of unique identifiers (e.g., “barcodes” or “barcode sequences”), with each type of unique identifier being present in multiple copies (e.g., approximately 200 to 400 barcode sequences) on the supramolecular structure 10. [00041] In practice, each type of unique identifier may include or be linked to a fluorescent molecule, such as a fluorescent molecule attached to a linker structure that chemically links to random or targeted (i.e., non-random) locations on the supramolecular structure 10). Each type of unique identifier may have a characteristic frequency at which it is excited or stimulated to emit detectable radiation (e.g., light) and/or a characteristic frequency at which it emits detectable radiation when excited. Monofunctional particles having a particular analyte affinity (i.e., an attached affinity binder for a respective analyte molecule, as discussed below) may therefore have a characteristic barcode sequence attached directly or indirectly (e.g., red, blue, yellow, or green, and so forth), a characteristic combination of fluorescent markers (each corresponding to a different barcode sequence) attached (e.g., red + blue, yellow + green, red + yellow + blue, and so forth), and/or a characteristic ratio of fluorescent markers attached (e.g., (2 red : 1 blue), (3 red : 2 green : 1 yellow). In this manner, detection at a read-out step of a characteristic frequency, combination of frequencies, or ratio of frequencies) may be used to determine and/or count the presence of monofunctional particles 100 bound to a respective analyte molecule.

[00042] Aspects of this are illustrated in FIG. 1 at step 16, at which it is illustrated that one or more types of barcode sequences 20 (e.g., barcode A (20A), barcode B (20B), barcode C (20C)) are linked to the supramolecular structure 10. Such linkage may be accomplished using complementary nucleic acid strand pairing or other complementary pairing, via covalent or other chemical bond formation, and/or by any other suitable attachment mechanism. By way of example, linkage of barcode sequences may be accomplished via the mechanism of linking to specific and known “staple” strands of nucleic acid used in the formation of scaffolded nucleic acid origami structures. In such an embodiment, because the sequence of such staple strands is known and because they occur in a fixed quantity and at fixed locations on the origami structure, different types of barcodes may be positioned on the supramolecular structure 10 at known and specific locations and numbers. By way of example, certain of the staples may react with or link to polymer linkers with specificity, where the polymer linkers correspond to different nucleic acid barcode sequences 20A, 20B, 20C, and so forth. That is, the nucleic acid polymer linkers may be the barcode sequences as described in the present examples. Due to the selectivity of the staples in terms of binding to specific locations on the scaffold, specific barcode sequences 20 may be targeted for attachment to respective staples which selectively bind to the scaffold at spatially separated locations to ensure the separation of the polymer linkers on the DNA origami.

[00043] Turning to FIG. 3, the polymer linkers corresponding to the barcode sequences 20 may in turn be complementary to respective fluorophores 104A, 104B, and 104C that comprise both a polymer strand complementary to one of the polymer linkers (e.g. barcode sequences 20A, 20B, and 20C respectively) and an attached fluorescent molecule characteristic of a respective set of fluorophores 104. Based on their complementary relationship a respective fluorophore 104 may bind to the complementary polymer linker 20 to form a respective barcode conjugate 108 A, 108B, 108C on the monofunctional particle 100. In practice, the fluorophores 104 may be bound to the barcode sequences 20 before, during or after binding of analyte molecules to the monofunctional particles 100.

[00044] In addition, as shown in FIG. 1, an affinity binder (or reactive group) linker structure 30 is linked (step 34) to the supramolecular structure 10, such as at a known location or binding site, e.g., a particular or targeted staple strand. The affinity binder linker 30 may be a nucleic acid structure or structures configured pair with the supramolecular structure 10, such as at a known or specified location on the supramolecular structure 10. In one embodiment at least a portion of the affinity binder linker 30 is also configured to form a complementary bond with an attachment portion of an affinity binder (or reactive group 40) to be attached (step 44) to the supramolecular structure 10 to form the monofunctional particle 100. As discussed herein, a respective analyte molecule may bind with specificity to the affinity binder 40 or reactive group of a monofunctional particle 100 through a chemical bond. In some embodiments, the affinity binder 40 or reactive group comprises a protein, a peptide, an antibody, an aptamer (RNA and DNA, a darpin, a polymer like PEG, or combinations thereof. [00045] The resulting monofunctional particle 100 has binding specificity to a specific analyte molecule, as determined by the affinity binder 40, and in the presence of the specific analyte molecule will bind to one such molecule. Monofunctional particles 100 that are not bound to analyte molecules maybe removed from the solution (using known separation techniques) or, in other implementations may remain in the solution. The solution containing monofunctional particles 100 bound to the analytes of interest may be processed, such as via flow cytometry, to determine the presence and/or quantity of the resulting analyte molecules, such as based upon the excitation and emissions of the barcodes on the monofunctional particles 100. In certain embodiments, as discussed herein, a given analyte of interest may be bound, such as at different regions or domains, by different affinity binders such that a complex of two or more different types of monofunctional particles 100 bind to a given analyte and the combination of the bound monofunctional particles 100 is indicate of an analyte molecule.

[00046] With reference to FIG. 1, it may be seen in this example that the linkage of the barcode sequences 20, affinity binder linker 30, and affinity binder 40 are depicted as separate and discrete steps from the synthesis (step 12) of the supramolecular structure 10. However, as may be appreciated from the preceding example, in practice one or more of these steps may be performed contemporaneous with, and intrinsic to, the synthesis of the supramolecular structure 10, such as in the context of synthesizing a scaffolded supramolecular structure using staples as described herein.

[00047] As noted herein, the example embodiment described above has a fixed signal strength determined by the number of unique identifiers (“barcodes”) bound to the surface of each monofunctional particle 100. In an alternative approach, separate and discrete barcode molecules or structures may not be attached to the supramolecular structure 10. Instead, the sequences (e.g., nucleic acid sequences) associated with the barcodes 20 may be incorporated onto a circular template suitable for rolling circle amplification, which may in turn be attached to the monofunctional particle 100. By incubating the circular template in solution with suitable transcription precursors and enzymes, a continuous strand of sequential barcode sequences 20 is generated so as to form a nanoball connected to the respective monofunctional particle 100. In this manner, the number of barcode sequences attached to the monofunctional particle 100 can be controlled by controlling the incubation environment (e.g., temperature, precursor concentrations or amounts, and so forth) and/or the duration of the incubation.

[00048] Turning to FIGS. 4-6, a process flow (FIG. 4) illustrating steps in the fabrication of one such embodiment of a monofunctional particle 100 is provided along with a corresponding visual representation (FIG. 5) of the circular template and of the process steps (FIG. 6). As shown in this example, a rolling circle amplification (RCA) template tag or polymer linker 110 is linked (step 114) to the supramolecular structure 10 (such as at a particular or targeted staple strand), such as via complementary sequence pairing or other suitable chemical bonding. The RCA template 120 (e.g., circular template) itself may then be linked (step 124) to the tag 110 via complementary sequence pairing or other suitable chemical bonding. In addition, as shown in FIG. 4 the affinity binder or reactive group linker 30 and affinity binder 40 or reactive group are linked (steps 34 and 44 respectively) to the supramolecular structure 10 as previously described (such as at a particular or targeted staple strand) to form the monofunctional particle 10. In practice, the supramolecular structure 10 may be selected, designed, or configured such that the respective linkage of the RCA template 120 and the affinity binder are spaced apart by a sufficient distance that the affinity binder is not interacted with or interfered with by the amplicon (e.g., nanoball) formed by the RCA template. By way of example, the supramolecular structure 10 may provide between approximately 5 nm to 1 pm separation between the RCA template linker 110 and the affinity binder linker 30. Though depicted as being on opposing ends of the supramolecular structure 10 in FIG. 6, the RCA template linker 110 and the affinity binder linker 30 may also be separated by selective placement on opposing surfaces of the supramolecular structure 10 (e.g., a DNA origami), such as by placement on a top surface and a bottom surface respectively.

[00049] With reference to FIGS. 4 and 6, it may be seen in this example that the linkage of the RCA template tag 110, RCA template 120, affinity binder linker 30, and affinity binder 40 are depicted as separate and discrete steps from the synthesis (step 12) of the supramolecular structure 10. However, as may be appreciated from the preceding example, in practice one or more of these steps may be performed contemporaneous with, and intrinsic to, the synthesis of the supramolecular structure 10, such as in the context of synthesizing a scaffolded supramolecular structure using staples as described herein.

[00050] An example of a RCA template 120 is shown in FIG. 5. In this example, the RCA template 120 comprises sequences (e.g., nucleic acid sequences) that may correspond to the unique identifiers or barcodes 20 of FIGS. 1 and 2. The barcode sequences incorporated in the circular template 120, however, are provided in a continuous or linked sequential arrangement and may consist of single or multiple copies of each barcode sequence 20. By way of example, by having different numbers of copies of each barcode sequence 20 in each circular template 120, a respective ratio of barcode signals may be generated using a given circular template, with the ratio conveying or characterizing the analyte molecule affinity for a given monofunctional particle 100 to which the circular template is attached.

[00051] In one example, the circular template 120 comprises a circularized singlestranded DNA (i.e., ssDNA) strand having a primer region 150 followed by a known or designed number of unique barcode region sequences (e.g., 20A, 20B, 20C, and so forth). Each barcode region sequence 20 may be separated a spacer sequence or region 152. Amplification of the RCA template 120 results in formation of an amplicon comprising the transcribed barcode sequences 20 in the order and number in which they are present on the RCA template 120 and, in one embodiment, causing the formation of a nanoball (see FIG. 5, nanoball 156) as described herein. In one non-limiting example, the nanoball 156 has a hydrodynamic radius of between approximately 100 nm to approximately 2 pm.

[00052] Association of respective complementary fluorophores (e.g., before, during, or after exposure to the analyte containing sample) causes a respective fluorescent molecule to be associated with each transcribed barcode sequence present in the nanoball 156. The duration of the incubation period over which the amplification is allowed to occur (as well as other reaction controlling characteristics), therefore, can directly determine the number of copies made of the RCA template 120, and therefore the resulting optical signal that may be associated with the nanoball 156. In this manner, measurable signal per analyte molecule may be increased or decreased based on the parameters of the detection mechanism so as to optimize the classification and/or quantification operation with respect to the analyte molecule. By way of example, in instances where the analyte molecule is at very low levels or concentrations, down to single-molecule quantification, a high-signal level per monofunctional particle 100 (and correspondingly per analyte molecule) may be useful. Conversely, in instances where the analyte-molecule is at relatively higher levels or concentrations, a lower-signal level per monofunctional particle 100 may be sufficient, or even desirable. In this manner, the detectable signal per monofunctional particle 100 can be customized or optimized as indicated by the use case or sample context. As noted above, this may provide a degree of signal optimization not available in embodiments corresponding to those described with respect to FIGS. 1 and 2, where the number of barcode sequences 20 per monofunctional particle 100 is fixed.

[00053] By way of further discussion, as noted herein the relative proportions of different barcode sequences (e.g., 20A, 20B, 20C, and so forth) may expressed in such an amplicon (such as a nanoball) and may be read out (such as via flow cytometry) and the results used to identify the analyte specificity of a respective monofunctional particle 100. This may allow a limited number of barcode sequences to, in various combinations, be used to identify a large number of analyte specificities. By way of example, a ratio of two barcode sequences A and B of 1 : 1 may correspond to a respective analyte specificity of a monofunctional particle 100. However, the relative proportion of each barcode sequence may be varied (e.g., 2: 1, 3: 1, 4: 1, 1 :2, 1 :3, 1 :4, and so forth) so as to create various unique measurable ratios, each corresponding to different analyte specificities while using only two barcode sequences. By increasing the number of barcode sequences employed, an even greater number of unique barcode sequence ratios may be achieved. For example, for barcode sequences A, B, and C, example ratios may include, but are not limited to: 1 : 1 : 1, 1 : 1 :2, 1 :2: 1, 2: 1 : 1, 1 : 1 :3, 1 :3: 1 :, 3: 1 : 1, 1 :2:3, 2: 1:3, 1 :3:2:, 2:3: 1, 3: 1 :2, 3:2: 1, and so forth. The number of permutations of ratios may be increased by adding additional numbers of available barcode sequences (e.g., D and E) such that the number of ratio permutations allows for a large number of unique ratios of barcode sequences each uniquely identifying an analyte specificity of respective monofunctional particles 100. Statistically, this may be represented as: (1) # permutations = (n — 2)³ where the number of permutations corresponds to the number of possible unique nanoballs based upon ratios of barcodes and n is the number of different barcode sections or sequences.

[00054] Turning to FIG. 7, an expanded view 180 of a length of the polymer sequence comprising nanoball 156, both before (left) and after (right) attachment of fluorescent molecules via complementary pairing, is provided. As shown by the expanded view, the strand from which the nanoball 156 is formed comprises multiple, linearly sequential copies of the barcode sequences 20 as specified by the RCA template 120. As shown in this example, the barcode sequences 20 encoded by the nanoball 156 are complementary to respective fluorophores 104A, 104B, and 104C that comprise both a polymer strand complementary to respective barcode sequences 20A, 20B, and 20C as well as an attached fluorescent molecule characteristics of a respective set of fluorophores 104. Based on their complementary relationship a respective fluorophore 104 may bind to the complementary barcode sequence 20 to form a respective barcode conjugate 108 A, 108B, 108C on the nanoball, and thereby on the monofunctional particle 100. In practice, the fluorophores 104 may be bound to the barcode sequences 20 before, during or after binding of analyte molecules to the monofunctional particles 100.

[00055] With the preceding in mind, an example of detection assay methodologies employing monofunctional particles 100 are described to provide a real -world context with respect to their use. As noted herein, in one implementation flow cytometry may be employed to evaluate a sample in which monofunctional particles 100 and analyte molecules of interest are present. In such a context, the flow cytometry apparatus may pass a solution containing the monofunctional particles 100 and analyte molecules through a tube or other passage. Within the tube, the solution is exposed to one or more excitation events (e.g., light pulses at wavelengths determined based upon the fluorophores 104 present on the monofunctional particles) and responsive emissions by the fluorophores 104 in response to the excitation events. Due to the flow mechanism and the constraints imposed by the analysis tube, data generation and collection is effectively one-dimensional (ID). Data generation and collection is also multi-channel in that emission data is generated and collected using multiple readout channels, each corresponding to a different emission spectrum. In practice, this may take the form of one readout channel per barcode sequence employed (i.e., 4 barcode sequences requires 4 readout channels, 8 barcode sequences requires 8 readout channels, and so forth).

[00056] Aspects of this approach are illustrated in FIGS. 8 and 9, in which a process flow (FIG. 8) illustrating steps in a flow cytometry assay is provided along with a corresponding visual representation (FIG. 9) of such a flow cytometry based assay. As shown in the figures, monofunctional particles 100 are added (step 210) to a solution 214 with a sample 206 comprising one or more analyte molecules 250 (FIG. 9) to be detected and/or quantified. As shown in FIG. 9, certain of the analyte molecules 250 for which corresponding affinity binders 40 are present in solution 214 are bound by their respective affinity binder 40, and thereby to the corresponding monofunctional particle(s) 100. In this manner complexes 260 (e.g., “sandwiches”) of monofunctional particles 100 and analyte molecules 250 may be formed. In practice, steric considerations may limit the number of monofunctional particles 100 that can bind to an analyte molecule 250, such as to two or three. In practice, monofunctional particles 100 having different fluorescent barcoding and different affinity binders 40 that are specific to different regions or domains of a given protein may create characteristic complexes 260 specific to the protein, as shown in FIG. 9.

[00057] The solution 214 may then undergo flow cytometry (step 220) during which the solution is exposed to one or more excitation wavelengths which cause emissions at characteristic wavelengths when fluorophores are present that are excitable by the excitation wavelengths. The resulting emission data 224 may be processed (step 228) to detect, classify, and/or quantify one or more analyte molecules 250 of interest. In particular, as shown in FIG. 8, the analyte data 232 is output by the step 228 and may be provided as an output to a user evaluating a sample for the analyte molecules 250 of interest and/or may be used in downstream processes for diagnosing a patient condition, treating a patient condition, and so forth. [00058] As noted above, identification and/or quantification of analyte molecules 250 of interest (e.g., proteins) may be based upon the detection of fluorescing complexes 260 using flow cytometry, where a complex 260 is known to form in the presence of protein of interest based upon the interaction of two different affinity binders 40 specific to the protein of interest. Conversely, detected fluorescence of non-complexed monofunctional particles 100 may be ignored or discarded in such a detection scenario based on complexes 260. By way of example, and turning to Table 1 below, a first monofunctional particle 100 having affinity for a first region or domain of a target protein may be characterized by measured fluorescence on readout channels 1, 2, and 3 (e.g., Channel 1 - 123, Channel 2 - 123, Channel 3 - 10) while a second monofunctional particle 100 having affinity for a second region or domain of the target protein may be characterized by measured fluorescence on readout channels 4, 5, and 6 (e.g., Channel 4 - 123, Channel 5 - 200, Channel 6 - 300). Observation by flow cytometry of the first or the second monofunctional particle in the absence of the other may be indicative of the absence of a binding event to the target protein. Conversely, observation by flow cytometry of both the first and the second monofunctional particle in proximity (i.e., as part of a complex 260) may be indicative of a binding event to the target protein. This example is illustrated in table form in Table 1 :

Table 1

[00059] While readout solutions based on a solution phase are one possible implementation, a surface-based detection implementation is illustrated by FIGS. 10 and 11, in which a process flow (FIG. 10) illustrating steps in a surface-based assay is provided along with a corresponding visual representation (FIG. 11) of such a surfacebased assay. As discussed in the preceding example and as shown in the figures, monofunctional particles 100 are added (step 210) to a solution 214 with a sample 206 comprising one or more analyte molecules 250 (FIG. 11) to be detected and/or quantified. As shown in FIG. 11, certain of the analyte molecules 250 for which corresponding affinity binders 40 are present in solution 214 are bound by their respective affinity binder 40, and thereby to the corresponding monofunctional parti cle(s) 100. In this manner complexes 260 (e.g., “sandwiches”) of monofunctional particles 100 and analyte molecules 250 may be formed. In practice, steric considerations may limit the number of monofunctional particles 100 that can bind to an analyte molecule 250, such as to two or three. In practice, monofunctional particles 100 having different fluorescent barcoding and different affinity binders 40 that are specific to different portions of a given protein may create characteristic complexes 260 specific to the protein, as shown in FIG. 11.

[00060] In the depicted example, the solution 214 may be flowed (step 280) over a substrate 290 (e.g., a read-out surface) to which the monofunctional particles 100 attach, such as via an anchor molecule(s) linked to the core structure of the supramolecular structure 10 of the monofunctional particle 100 as discussed herein. By way of example, the anchor molecule of a monofunctional particle may comprise a nucleic acid strand complementary to a strand or strands provided on the substrate 290, though any suitable chemical attachment or bonding mechanism may be utilized. Depending on the implementation, attachment of the monofunctional particles 100 may be random with respect to the surface or may be at defined locations defined on the surface (e.g., “landing pads” or wells).

[00061] The substrate 290, once populated by monofunctional particles 100 and complexes 260 may then be processed to generate emission data 224, such as in response to exposure to excitation wavelengths of radiation. For example, the populated substrate 290 may be exposed to one or more excitation wavelengths which cause emissions at characteristic wavelengths when fluorophores are present that are excitable by the excitation wavelengths. The resulting emission data 224 may be processed (step 228) to detect, classify, and/or quantify one or more analyte molecules 250 of interest. In particular, as shown in FIG. 8, the analyte data 232 is output by the step 228 and may be provided as an output to a user evaluating a sample for the analyte molecules 250 of interest and/or may be used in downstream processes for diagnosing a patient condition, treating a patient condition, and so forth.

[00062] Unlike the flow cytometry example discussed herein, which corresponds in practice to a one-dimensional data acquisition, the substrate-based approach described herein may allow for data collection in an area corresponding to the substrate 290 (i.e., two-dimensional (2D) data acquisition) and may be suitable for 2D-based data acquisitions, such as image-based acquisitions. Depending on the context, such imagebased approaches at step 284 may be beneficial.

[00063] With this in mind, identification and/or quantification of analyte molecules 250 of interest (e.g., proteins) may be based upon the detection of fluorescing complexes 260 using monofunctional particles attached to a substrate 290, where a complex 260 is known to form in the presence of protein of interest based upon the interaction of two different affinity binders 40 specific to the protein of interest. Conversely, detected fluorescence of non-complexed monofunctional particles 100 may be ignored or discarded in such a detection scenario based on complexes 260. By way of example, a complex 260 emitting across a range of channels (e.g., channels 1-6) known to be associated with a respective target analyte of interest can be characterized and localized (e.g., based on Cartesian x, y coordinates) so that a count of one analyte molecule interest at the respective coordinates can be made. Conversely, emission data that is incomplete across the range of channels (e.g., for only 3 of 6 channels) can be indicative of the absence of a complex 260 (e.g., a single monofunctional particle 100) and, therefore, the absence of a binding event to the target protein. [00064] As may be appreciated, aspects of the presently described structures and techniques may be implemented or used if the further context of a device or system, such as an analyte detection or quantification system 320 as shown in FIG. 12. In particular, FIG. 12 shows an analyte detection or quantification system 320 that includes a controller 324. The controller 324 includes processor 328 and a memory 332 storing instructions configured to be executed by the processor 328. The controller 324 includes a user interface 336 and communication circuitry 340, e.g., to facilitate communication over the internet 350 and/or over a wireless or wired network. The user interface 336 facilitates user interaction with operational results or parameter specification as provided herein.

[00065] The processor 328 is programmed to receive data and execute operational commands for performing one or more operations as described herein, such as an analyte molecule identification and/or quantification operation using a fluorescence-based imaging system or flow cytometer. With this in mind, the system 300 also includes a detection component 360, such as an area imaging component and/or flow cytometer that operates to control operations on or involving monofunctional particles 100 as may be used to identify or count analyte molecules as discussed herein. An excitation and/or emission readout controller 364 may be present that controls excitation operations and/or emission readout operations performed on monofunctional particles 100, and so forth at appropriate time points during a detection operation. A sensor 368 may be provided as one or more of an optical sensor (e.g., a fluorescent sensor, an infrared sensor), an image sensor, an electrical sensor, or a magnetic sensor for detecting suitable data generated by or at the monofunctional particle 100.

[00066] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. An analyte quantification system, comprising: a detection component comprising: an excitation and emission controller configured to stimulate emissions by one or more monofunctional particles when present in a detection area; one or more sensors configured to detect emissions from the one or more monofunctional particles when present in the detection area; a controller in communication with the detection component, wherein the controller is configured to quantify one or more analytes of interest based on detected emissions from the one or more monofunctional particles, wherein each monofunctional particle has an affinity for a single analyte, is capable of binding to only a single copy of the analyte to which it has affinity, and emits emissions in a pattern indicative of the affinity of the monofunctional particle.

2. The analyte quantification system of claim 1, wherein the detection component comprises a flow cytometer and the detection area comprises a flow tube of the flow cytometer.

3. The analyte quantification system of claim 1, wherein the detection component comprises an area imager configured to image a substrate on which the one or more monofunctional particles are bound.

4. The analyte quantification system of claim 1, wherein the controller is configured to quantify the one or more analytes of interest based on detected emissions from pairs of monofunctional particles bound to single analyte molecules via different respective affinity binders, such that the combined emissions of a respective pair of monofunctional particles in proximity is indicative of one count of the analyte molecule.

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5. A monofunctional particle, comprising: a nucleic acid supramol ecul ar structure; an affinity binder linker; an affinity binder linked to the affinity binder linker and having a binding affinity for a specific analyte; and a plurality of copies of one or more barcode sequences, wherein the one or more barcode sequences, in combination, are indicative of the affinity binder linked to the monofunctional particle.

6. The monofunctional particle of claim 5, wherein the nucleic acid supramolecular structure comprises a deoxyribonucleic acid (DNA) origami, a ribonucleic acid (RNA) origami, a hybrid DNA/RNA origami, a single-stranded DNA origami, a single-stranded RNA origami, a hierarchically composed DNA and/or RNA origami, enzymatically synthesized nucleic acid structures, or structures created by nucleic acid tile assembly.

7. The monofunctional particle of claim 5, wherein the nucleic acid supramolecular structure is scaffolded with one or more scaffolds.

8. The monofunctional particle of claim 5, wherein the nucleic acid supramolecular structure comprises a prescribed two-dimensional (2D) or three-dimensional (3D) shape.

9. The monofunctional particle of claim 5, wherein the specific analyte comprises a specific protein.

10. The monofunctional particle of claim 5 further comprising an anchor molecule configured to immobilize the monofunctional particle on a substrate.

11. The monofunctional particle of claim 5, wherein the one or more barcode sequences comprise two, three, or four barcode sequences.

12. The monofunctional particle of claim 5, wherein the plurality of copies of the barcode sequences taken together comprise a characteristic ratio of each barcode sequence that is indicative of the affinity binder linked to the monofunctional particle.

13. The monofunctional particle of claim 5, further comprising a respective and corresponding fluorophore bound by complementary pairing to each copy of a respective barcode sequence.

13. A monofunctional particle, comprising: a nucleic acid supramol ecul ar structure; an affinity binder linker; an affinity binder linked to the affinity binder linker and having a binding affinity for a specific analyte; a rolling circle amplification (RCA) tag; and a RCA template linked to the RCA tag and comprising a plurality of copies of two or more barcode sequences, wherein the two or more barcode sequences, in combination, are indicative of the affinity binder linked to the monofunctional particle.

14. The monofunctional particle of claim 13, wherein the nucleic acid supramolecular structure comprises a deoxyribonucleic acid (DNA) origami, a ribonucleic acid (RNA) origami, a hybrid DNA/RNA origami, a single-stranded DNA origami, a single-stranded RNA origami, a hierarchically composed DNA and/or RNA origami, enzymatically synthesized nucleic acid structures, or structures created by nucleic acid tile assembly.

15. The monofunctional particle of claim 13, wherein the nucleic acid supramolecular structure is scaffolded with one or more scaffolds.

16. The monofunctional particle of claim 13, wherein the nucleic acid supramolecular structure comprises a prescribed two-dimensional (2D) or three-dimensional (3D) shape.

17. The monofunctional particle of claim 13, wherein the specific analyte comprises a specific protein.

18. The monofunctional particle of claim 13 further comprising an anchor molecule configured to immobilize the monofunctional particle on a substrate.

19. The monofunctional particle of claim 13, wherein the two or more barcode sequences comprise two, three, or four barcode sequences.

20. The monofunctional particle of claim 13, wherein the plurality of copies of the barcode sequences taken together comprise a characteristic ratio of each barcode sequence that is indicative of the affinity binder linked to the monofunctional particle.

21. The monofunctional particle of claim 13, further comprising a nanoball formed as an amplicon of the RCA template formed as a continuous strand comprising a repeated and sequential sequence of the plurality of copies of the two or more barcode sequences.

22. The monofunctional particle of claim 21, further comprising a respective and corresponding fluorophore bound by complementary pairing to each copy of a respective barcode sequence of the nanoball.

23. A method for forming a monofunctional particle, the method comprising the steps of: synthesizing or acquiring a nucleic acid supramolecular structure; attaching an affinity binder linker to the nucleic acid supramolecular structure; attaching an affinity binder to the affinity binder linker, wherein the affinity binder has a binding affinity for a specific analyte; and attaching multiple copies of one or more barcode sequences to the nucleic acid supramolecular structure, wherein the one or more barcode sequences, in combination, are indicative of the affinity binder linked to the monofunctional particle.

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24. The method of claim 23, wherein the one or more barcode sequences comprise two, three, or four barcode sequences.

25. The method of claim 23, wherein the plurality of copies of the barcode sequences taken together comprise a characteristic ratio of each barcode sequence that is indicative of the affinity binder linked to the monofunctional particle.

26. The method of claim 23, further comprising attaching a respective and corresponding fluorophore bound by complementary pairing to each copy of a respective barcode sequence.

27. A method for forming a monofunctional particle, the method comprising the steps of: synthesizing or acquiring a nucleic acid supramolecular structure; attaching an affinity binder linker to the nucleic acid supramolecular structure; attaching an affinity binder to the affinity binder linker, wherein the affinity binder has a binding affinity for a specific analyte; and attaching a rolling circle amplification (RCA) tag to the nucleic acid supramolecular structure; and attaching a RCA template to the RCA tag, wherein the RCA template comprises a plurality of copies of two or more barcode sequences, wherein the two or more barcode sequences, in combination, are indicative of the affinity binder linked to the monofunctional particle.

28. The method of claim 27, wherein the plurality of copies of the barcode sequences taken together comprise a characteristic ratio of each barcode sequence that is indicative of the affinity binder linked to the monofunctional particle.

-SO-

29. The method of claim 27, further comprising forming a nanoball as an amplicon of the RCA template, wherein the amplicon is formed as a continuous strand comprising a repeated and sequential sequence of the plurality of copies of the two or more barcode sequences.

30. The method of claim 29, further comprising associating a respective and corresponding fluorophore by complementary pairing to each copy of a respective barcode sequence of the nanoball.

31. A method for quantifying an analyte of interest in a sample, the method comprising: adding a sample comprising the analyte of interest and a plurality of monofunctional particles to a solution, wherein some or all of the monofunctional particles have an affinity for the analyte of interest and each monofunctional particle, when excited, emits emissions in a pattern indicative of an affinity binder attached to the respective monofunctional particle; performing flow cytometry on the solution; generating emission data as a result of the flow cytometry; and quantifying the analyte of interest within the sample based on the emission data.

32. The method of claim 31, wherein each monofunctional particle comprises: a nucleic acid supramol ecul ar structure; an affinity binder linker; the affinity binder linked to the affinity binder linker and having a binding affinity for a specific analyte; and a plurality of barcode sequences, wherein the one or more barcode sequences, in combination, are indicative of the affinity binder linked to the monofunctional particle.

33. The method of claim 32, wherein the plurality of barcode sequences taken together comprise a characteristic ratio of each barcode sequence that is indicative of the affinity binder linked to the monofunctional particle.

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34. The method of claim 32, further comprising a respective and corresponding fluorophore bound by complementary pairing to each copy of a respective barcode sequence.

35. The method of claim 31, wherein quantifying the analyte of interest is based on detected emissions from pairs of monofunctional particles bound to single molecules of the analyte of interest via different respective affinity binders, such that the combined emissions of a respective pair of monofunctional particles in proximity is indicative of one count of the analyte molecule.

36. A method for quantifying an analyte of interest in a sample, the method comprising: adding a sample comprising the analyte of interest and a plurality of monofunctional particles to a solution, wherein some or all of the monofunctional particles have an affinity for the analyte of interest and each monofunctional particle, when excited, emits emissions in a pattern indicative of an affinity binder attached to the respective monofunctional particle; flowing the solution over a substrate surface, wherein the plurality of monofunctional particles attach to the substrate surface; acquiring image data of the substrate surface in the presence of a plurality of excitation wavelengths; generating emission data in response to the excitation wavelengths; and quantifying the analyte of interest within the sample based on the emission data.

37. The method of claim 36, wherein each monofunctional particle comprises: a nucleic acid supramol ecul ar structure; an affinity binder linker; the affinity binder linked to the affinity binder linker and having a binding affinity for a specific analyte; and

-32- a plurality of barcode sequences, wherein the one or more barcode sequences, in combination, are indicative of the affinity binder linked to the monofunctional particle.

38. The method of claim 37, wherein the plurality of barcode sequences taken together comprise a characteristic ratio of each barcode sequence that is indicative of the affinity binder linked to the monofunctional particle.

39. The method of claim 37, further comprising a respective and corresponding fluorophore bound by complementary pairing to each copy of a respective barcode sequence.

40. The method of claim 37, further comprising an anchor molecule configured to bind to substrate surface to attach the respective monofunctional particle to the substrate surface.

41. The method of claim 36, wherein quantifying the analyte of interest is based on detected emissions from pairs of monofunctional particles bound to single molecules of the analyte of interest via different respective affinity binders, such that the combined emissions of a respective pair of monofunctional particles in proximity is indicative of one count of the analyte molecule.

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