WO2025054146A2

WO2025054146A2 - Label free analyte binder and labeled probe

Info

Publication number: WO2025054146A2
Application number: PCT/US2024/045094
Authority: WO
Inventors: Min-Jui Richard Shen; Denis MALYSHEV
Original assignee: Eglint Inc
Current assignee: Eglint Inc
Priority date: 2023-09-08
Filing date: 2024-09-04
Publication date: 2025-03-13
Anticipated expiration: 2026-03-08
Also published as: WO2025054146A3

Abstract

Analyte binding to analyte binding moieties such as oligos on an array such as a surface is disclosed. Binding is assayed using, for example, fluorophore labeled probes rather than using fluorophores on the analyte binding moieties, resulting in a substantial increase in system efficiency.

Description

LABEL FREE ANALYTE BINDER AND LABELED PROBE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This document claims the benefit of priority to US Prov Ser No 63/581,326, filed September 8, 2023, the contents of which are hereby incorporated by reference in their entirety. BACKGROUND

[0002] High throughput detection of large numbers of analytes simultaneously in a sample remains challenging, both due to the technical difficulties of analyte identification and due to the cost of synthesizing systems capable of detecting high numbers of analytes accurately and repeatably.

SUMMARY

[0003] Disclosed herein are systems, methods, and compositions for efficient high throughput analyte detection. Some such systems allow the detection of any number of analytes across a broad range of concentrations using unlabeled analyte-binding oligos in solution or tethered to a surface, and a labeled universal probe sufficient to assay for the presence of a binding configuration in the analyte-biding oligos. The systems herein allow for detection of specific analytes across a broad range of concentrations using a common labeled fluorophore that does not need to be unique or specific to any of the analytes to be detected.

[0004] Consistent with the above, disclosed herein are systems for detecting an analyte, said systems comprising one or more of a first oligonucleotide comprising an analyte specific binding region bounded by a first universal region and in some embodiments a second universal region; a second oligonucleotide segment comprising a first universal region reverse complement region and a paired fluorophore; and in some embodiments a third oligonucleotide segment comprising a second universal region reverse complement region and a paired fluorophore complement.

[0005] Also disclosed herein are methods of high throughput sample analysis, said methods comprising one or more of contacting a sample to a plurality of analyte-specific oligos that share common universal detection oligo binding sites such as single part or bipartite oligo binding sites, and assaying for binding of at least one of the plurality of analyte-specific oligos to its analyte by contacting a plurality of analyte-specific oligos to a universal detection oligo population.

[0006] Also disclosed herein are surfaces, such as those comprising one or more of a first oligo comprising a first analyte binding region and a first adapter and a second adapter, wherein the first oligo is tethered to the surface and bound to a first analyte such that the first adapter and the second adapter are in local proximity, and wherein the first oligo is bound to a first adapter binding region and a second adapter biding region, such that the first adapter biding region and the second adapter binding region are held in proximity.

[0007] Similarly, disclosed herein are surface comprising one or more of unlabeled specific analyte probes, and capable of being bound at the unlabeled specific analyte probes to labeled nonspecific adapter probes, wherein binding of at least one of the unspecific analyte probes causes reconfiguration of at least one of the labeled nonspecific adapter probes such that a label of the at least one of the labeled nonspecific adapter probes may be detected.

[0008] Also disclosed herein are methods of detecting a plurality of analytes without requiring covalently labeled analyte probes, said methods comprising one or more of contacting a sample to a plurality of analyte probes, wherein the analyte probes undergo a conformational change to an analyte binding configuration in response to binding to their respective analytes in the sample, and binding the plurality of analyte probes to a population of universal labeled probes, wherein the universal labeled probes emit a signal only when the analyte probes are in an analyte binding configuration.

[0009] Some aspects of the disclosed methods and systems above share as a common feature that the specificity conveying oligos or other moieties, such as the analyte binding agents, are not covalently labeled with a fluorophore or other detection moiety. Similarly, some aspects of the disclosed methods and systems above share as a common feature that the fluorophore or otherwise labeled component does not convey specificity or otherwise bind to or is not designed to bind to any analyte to be detected. Similarly, some aspects of the disclosed methods and systems above share as a common feature that the fluorophore or otherwise labeled component may be drawn from a re-usable pool, such that the same reagent, or aliquots of the same reagent may be used across multiple analytes, or multiple experiments each assaying multiple reagents. [0010] Some aspects of the disclosed methods and systems above share as a common feature that analytes are detected based on the location on a solid surface where fluorescence is observed from a probe, such as a nonspecific probe applied generally to the surface and in some cases drawn from a common pool from which aliquots are drawn across multiple assays.

INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1 depicts a probe labeled with a split reporter comprising a quencher BHQ1 and a fluorophore FAM bound to an unlabeled analyte-binding oligo complexed to its protein target. [0013] Fig. 2 depicts a probe labeled with the FAM half of a fluorophore-quencher pair and having an DBCO immobilization tag, bound to an analyte-binding oligo complexed to its protein target and harboring the fluorophore-quencher pair complement BHQ1.

[0014] Fig. 3 presents a control assay having a split fluorophore-quencher system attached to the analyte-binding oligo.

[0015] Fig. 4 presents a assay having a split fluorophore-quencher system attached to the probe. [0016] Fig. 5 presents quantification (distance measurements) for the assays of Fig. 3 and Fig. 4. DETAILED DESCRIPTION

[0017] Disclosed herein are systems, devices, compositions, and methods for the rapid detection of a large number of target analytes across a broad range of analyte concentrations in a high throughput system. Although the technology herein is compatible with antibodies and other protein-based detection moieties, some preferred embodiments rely upon target-specific aptamers that are readily synthesized using low-cost nucleic acid synthesis approaches and delivered to a surface of a chip, flow cell or bead using well-established chemical approaches. Clusters may be spotted onto surfaces or deposited into wells via beads or directly in solution at a very high density, such that a large number of analytes may be assayed for in a single reaction. Assays comprise binding using a fluorophore-labeled or otherwise labeled probe that binds to conserved regions of the analyte-binding probes, such that a uniform labeled probe set.

[0018] Assays are effected by observing the effect of two changes on the analyte-binding oligos or other affinity reagents: firstly, a change in stability resulting from binding to target analytes, and secondly, a counteracting challenge to stability resulting from an environmental change such as an increase in temperature or other environmental challenge mentioned herein or known in the art - examples bring changes in buffer, ion concentration, or other condition that may impact oligonucleotide conformation. These two changes interact such that a reporter of aptamer or other affinity reagent conformation such as a paired fluorophore system on a probe that binds conserved regions of the analyte-binding oligos will often be stabilized upon target analyte binding. Consequently, the challenge presented by an environmental change will impact the output of such a reporter only when administered at a larger magnitude than that administered to an unbound affinity reagent. Alternatives where target analyte binding is detrimental to signaling output are also contemplated.

[0019] By gradually or incrementally increasing the magnitude of an environmental change (such as an addition of heat or a change in ion concentration), one can observe (or compare to previously calculated values for) the temperature or other condition at which a change in fluorescence or other reporter occurs, both for bound and unbound affinity reagents. By observing a change in persistence of the fluorescence or other reporter relative to an observed or known unbound control upon incremental increases in the environmental parameter, one can infer binding of the target analyte to the affinity reagent.

[0020] Alternately, by selecting affinity reagents having known changes in persistence upon binding to target reagents, one can subject a set of affinity reagents to a temperature where bound and unbound affinity reagents are predicted to differentially fluoresce depending upon target analyte binding. Using this approach, one may rapidly assay for a number of target analytes using a simple environmental change regimen of one, two or three conditions.

[0021] Notably, target analyte detection is a function of the environmental condition and its impact upon the affinity reagent fluorescence. The chemistry of the target analytes is relevant only so long as the affinity reagents are able to bind them. As aptamers, for example, can be synthesized to bind to a broad range of target molecules, one can detect a broad range of target analytes using a relatively universal environmental change/reporter assay.

[0022] Accordingly, at little cost and with little time in reagent synthesis, using a common reporter assay, in some cases a common reporter probe and a common environmental change as a challenge, one may assay for a broad range of biochemically diverse target analytes, across a broad range of concentrations, in a single, rapidly executed and easily measured assay.

[0023] Systems, methods, and compositions may share one or more of the common features below.

[0024] Analyte-binding oligos or other analyte binders. Systems, compositions and methods herein often employ an analyte binder or an array or analyte binder so as to detect analytes in a sample. Exemplary analyte binders are oligos, though other analyte binding moieties are consistent with the disclosure herein.

[0025] Common features of the analyte binders in that they comprise two distinct elements. Firstly, analyte binders comprise an analyte binding region. The analyte binding region is specific to a particular analyte or category of analyte and its configuration is sensitive to or changes in response to analyte presence or analyte binding. Secondly, analyte binders comprise one or more than one configuration-reporting moieties that are often common to some or all of the analyte binders in an assay.

[0026] The analyte binding region interacts directly with the analyte in a manner that is impacted by binding to the analyte, such that binding to the analyte alters the configuration of the analyte binding region. The binding is in many cases temperature sensitive, such that temperature elevation or in some cases temperature decrease may abolish analyte binding. Similarly, some temperatures or ranges of temperatures may trigger analyte binding regions to contract or to assume a tighter configuration that may mimic analyte binding. Accordingly, in some cases analyte binding may manifest itself as preservation or early abolition of a tight configuration of an analyte binding region.

[0027] Exemplary analyte binding regions are oligos such as aptamer oligos.

[0028] Although the technology herein is compatible with antibodies and other protein-based detection moieties, some preferred embodiments rely upon target-specific aptamers that are readily synthesized using low-cost nucleic acid synthesis approaches and delivered to a surface of a chip, flow cell or bead using well-established chemical approaches. Clusters may be spotted onto surfaces or deposited into wells via beads or directly in solution at a very high density, such that a large number of analytes may be assayed for in a single reaction.

[0029] Assays are carried out by observing the effect of two changes on the aptamers or other affinity reagents: firstly, a change in stability resulting from binding to target analytes, and secondly, a counteracting challenge to stability resulting from an environmental change such as an increase in temperature or other environmental challenge mentioned herein. These two changes interact such that a reporter of aptamer or other affinity reagent conformation such as a paired fluorophore system will often be stabilized upon target analyte binding. Consequently, the challenge presented by an environmental change will impact the output of such a reporter only when administered at a larger magnitude than that administered to an unbound affinity reagent.

[0030] Aptamers may be designed to target specific analytes or classes of analytes or binding partners using a number of distinct approaches. In some embodiments, the binding is specific binding for a target molecule, such target molecule having a three-dimensional chemical structure, other than a polynucleotide, that binds to the aptamer through a mechanism which is predominantly independent of Watson/Crick base pairing or triple helix binding. In some embodiments, the aptamer can be one such as those described in S. Lapa, et al., Molecular Biotechnology volume 58, p. 79-92 (2016); or S. Gao, et al., Analytical and Bioanalytical Chemistry volume 408, p. 4567-4573 (2016)), herein incorporated by reference.

[0031] Beneficially, aptamers can be developed using any of a number of established SELEX methods (Systematic Evolution of Ligands by Exponential Enrichment) (Tuerk et al., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science (New York, NY). 1990;249(4968):505-10; Ellington et al., In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346(6287):818-22; Robertson et al., Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature. 1990;344(6265):467-8; Lee et al., Aptamer therapeutics advance. Curr Opin Chem Biol. 2006;10(3):282-9; Banerjee et al., Aptamers: multifunctional molecules for biomedical research. J Mol Med (Berl). 2013;91(12): 1333-42; Hong et al., Single-stranded DNA aptamers against pathogens and toxins: identification and biosensing applications. Biomed Res Int. 2015;2015 :419318; Zhang et al., Practical application of aptamer-based biosensors in detection of low molecular weight pollutants in water sources. Molecules (Basel, Switzerland), 2018;23(2):344). Each of these references is incorporated by reference in its entirety. Third- party synthesized or designed aptamers can also be purchased commercially. Once discovered, aptamers can be synthesized by solid phase synthesis with modifiers such that dye pairs can be included in the aptamer sequence or connected to a modified nucleotide at specific locations that result in different emission properties in the binding competent and binding incompetent states. Such modifiers can be commercially obtained (e.g., IDT DNA, LGC Biosearch Technologies, or Trilink, Inc. (a subsidiary of Miravai Life Sciences (California) at time of filing).

[0032] Aptamers include nucleic acids that are identified from a candidate mixture of nucleic acids by the process referred to as SELEX and variations thereof. In some embodiments, the aptamer is developed by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture may be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby aptamers to the target molecule are identified. Affinity interactions may vary in degree; however, in this context, the "specific binding affinity" of an aptamer for its target means that the aptamer binds to its target generally with a higher degree of affinity than it may binds to other, non-target, components in a mixture or sample. A "cluster of aptamers" is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence and are grouped together in a defined location on a surface. An aptamer can include any suitable number of nucleotides. Different aptamers may have either the same number or a different number of nucleotides. Aptamers may comprise or be DNA or RNA or variants thereof, and may comprise single stranded, double stranded, and/or hairpin regions.

[0033] In some embodiments, the nucleic acid composition of an aptamer can be varied to produce an aptamer with a selected persistence upon self-folding into a binding competent state or to optimize its affinity for its target. In some embodiments, the modified nucleic acid in the aptamer can include or exclude: peptide-nucleic acids (PNA), locked nucleic acids (LNA), or normal ribonucleic (RNA) and deoxyribonucleic (DNA) acids. PNAs have a peptide-backbone rather than a ribose-phosphate backbone of normal DNA. The PNA backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The purine and pyrimidine bases are linked to the PNA backbone by a methylene bridge (-CH2-) and a carbonyl group (- (C=O)-), The PNA backbone thus lacks charged phosphate groups. PNAs are not easily recognized by either native nucleases or proteases, imbuing them resistance to enzymatic degradation and pH stability. The LNA backbone comprises a ribose moiety which is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon locking the ribose in the 3-endo (North) conformation. The locked ribose conformation enhances base stacking and backbone pre-organization and significantly increases the duplex stability of LNA/DNA duplexes. Methyl phosphonate backbones replace the charged anionic phosphate with a neutral methyl phosphonate ester. Thiophosphonate backbones comprise non-bridging oxygen on the phosphate backbone to form a phosphorothioate (PS) linkage. Thiophosphonate backbones exhibit nuclease resistance should a nuclease be present in the sample. Not all of these constituent nucleotides can be included in SELEX, but can be used to modify a nucleic acid molecule.

[0034] The aptamer can in some cases further comprise one or a plurality of non-natural nucleotides, such as iso-G or iso-C (or derivatives thereof), as described in Richert, C., et al. J. Am. Chem. Soc. 118, 4518-4531 (1996), herein incorporated by reference. Exemplary nonnatural nucleotides include diflurotoluene (or derivatives thereof), as described in Schweitzer, B. A., et al., J. Am. Chem. Soc. 117, 1863-1872 (1995), herein incorporated by reference; MM02 or SICS (or derivatives thereof), as described in Leconte, A. M. et al. J. Am. Chem. Soc. 130, 2336-2343 (2008), herein incorporated by reference; Ds or Diol-Px (or derivatives thereof), as described in Yamashige, R. et al. Nucl. Acids Res. 40, 2793-2806 (2012), herein incorporated by reference; P or Z (or derivatives thereof), as described in Yang; Z., et al., J. Am. Chem. Soc. 133, 15105-15112(2011), herein incorporated by reference; NaM or 5SICS (or derivatives thereof), as described in Malyshev, D. A. et al. Proc. Natl Acad. Sci. USA 109, 12005-12010 (2012), herein incorporated by reference; or of an expanded genetic code as described in Kimoto et al., Chem. Soc. Rev., 2020,49, 7602-7626, herein incorporated by reference. In some embodiments, the aptamers comprising one or a plurality of non-natural nucleotides can be modified before, or after SELEX identification so as to include a non-natural nucleotide.

[0035] In some embodiments, the nucleotides include SELEX-compatible nucleotides (or a variant thereof) or that can be introduced into a nucleic acid. In some embodiments, the SELEX- compatible nucleotides can include or exclude the following nucleotide modifications: substitution of 2'-OH by fluor (F), modification of 2'-OH by a methyl group (CH3), substitution of 2'-OH by an amino group (NH2), a Locked Nucleic Acid (LNA) with methylene bridge between 2'-0 and 4'-C, modification of C-5 by Bromine (Br), modification of C-5 by Iodine (I), and substitution of 4-0 by Sulfur (S). In some embodiments, the SELEX-compatible nucleotides can include or exclude: 2'-Fluoro-dUTP. 2'-Fluoro-dCTP, 2'-Fluoro-dATP, 2'-Fluoro-dGTP, 2'- Fluoro-dNTP, 2'OMe-UTP, 2'OMe-CTP, 2'OMe-ATP, 2'OMe-GTP, 2'NH2-dUTP, 2'NH2- dCTP, 2'NH2-dATP, 2'NH2-dGTP, LNA-ATP, LNA-GTP, LNA-CTP, LNA-UTP, 5-Bromo- dUTP, 5-Iodo-UTP, 4-Thio-UTP, s4UTP, and 4sUTP. In some embodiments, the SELEX- compatible nucleotides can be those described in Komarova et al., Molecules. 2019 Oct; 24(19): 3598, herein incorporated by reference. A SELEX-compatible nucleotide can be a nucleotide which can be included in a SELEX process without negatively impacting the ability of the SELEX process to arrive at an aptamer which can selectively bind to a target.

[0036] Unlike the analyte binding regions, the configuration-reporting moiety or moieties, or common single or bipartite universal detection oligo binding sites, of the analyte-binding oligos or other analyte binders are common to some or all of an analyte binder population.

Configuration-reporting moieties are generally oligos (usually one or two) tethered to the analyte-biding regions, often at one or both ends of the analyte binding regions. Configuration reporting moieties are generally not sensitive in their structure to analyte binding. However, their proximity to one another, or the proximity of a single reporting moiety to the analyte binding region and its reporter pair molecule, is sufficient for analyte detection.

[0037] Split reporter activity is sensitive to analyte binding moiety configuration, such that analyte-binding oligos, for example, may draw the configuration reporting moieties into proximity to one another that is closer or farther as a function of analyte-binding oligo configuration, which is in turn a function in part of analyte binding.

[0038] Thus, configuration-reporting moieties serve to report the presence of an analyte not through analyte binding but through their proximity to one another, which is itself a function of analyte binding.

[0039] A depiction of this configuration is shown in Fig. 1.

[0040] In a variant on this core approach, some analyte-binding oligos or other analyte binders have only a first configuration moiety, often at one end of the analyte-binding oligo or other binding moiety. At the other end of the analyte-binding oligo or other binding moiety is found one half of a split signaling moiety, such as a fluorophore or a quencher.

[0041] The first configuration moiety recruits a probe to the analyte-binding oligo or other binding moiety in a configuration such that the second half of the split signaling moiety, such as a quencher or a fluorophore, is in proximity to the first half of a split signaling moiety in an analyte-binding dependent manner. That is, only when the analyte-binding oligo or other binding moiety is in a closed, tight or analyte bound configuration is the first half of the signaling pair of the analyte-biding oligo or other analyte binding moiety in proximity to the second half of the analyte binding pair on the probe. Conveniently, the first half of the signaling pair of the analyte-biding oligo or other analyte binding moiety is often tethered to the 5’ end of a primer used in synthesizing the analyte-biding oligo or other binding moiety, so as to facilitate its attachment, though other biochemical approaches for signaling moiety attachment and other positions of attachment are also consistent with the disclosure herein.

[0042] A depiction of this configuration is shown in Fig. 2.

[0043] Some configuration-reporting moieties serve as binding sites for a first probe segment, or of a first probe segment and a second probe segment as discussed below.

[0044] One or more configuration-reporting moieties are in some cases added onto analytebinding oligos or other analyte binders subsequent to their selection. Oligos may be impacted in their binding properties by addition of 5’ and 3’ end adapters, for example in the strength of binding or in the temperature at which binding dissociates, so in some cases oligo binding to target analytes is confirmed or reevaluated subsequent to addition of the configuration-reporting moieties.

[0045] Alternately, in some cases configuration-reporting moieties are present on analytebinding oligos or other analyte binders concurrently with their selection. For example, SELEX often comprises polymerase chain reaction amplification of potential analyte binding segments bound by primer binding sites. These primer binding sites may in some cases serve as configuration-reporting moieties for some or all of the analyte-binding oligos or other analyte binders in a given array.

[0046] Labeled probes. Systems, compositions and methods herein often employ a labeled probe system. Probes herein generally comprise a bipartite reporter such as a split fluorophore or a fluorophore-quencher pair that is proximity-sensitive. That is, probe signaling is a function of proximity of a first part and a second part of the probe reporter system, as seen in Fig. 1.

[0047] Alternately, some labeled probe systems employ a labeled probe harboring one half of a split bipartite reporter such as a split fluorophore or a fluorophore-quencher pair that is proximity-sensitive. In these systems, half of a split probe is located on the probe while the remaining half is attached to the analyte-binding probe or other analyte binding moiety, such that reporter signaling is a function of proximity of a first part and a second part of the split reporter system, wherein the first part and the second part are localized on functionally distinct components as seen in Fig. 2.

[0048] First part and second part proximity results in a change in a detectable signal or capacity to generate or yield a detectable signal, such that detection of a signal or a change in signal is indicative of the proximity of the first part and the second part of the reporter system. In various embodiments, proximity of the first part and the second part of the reporter system results in gaining capacity to generate a signal, quenching or loss of capacity to generate a signal, or a change in signal strength, wavelength, or any other detectable feature of the reporter system. [0049] The first part and a second part of the probe reporter system are in some cases tethered by a common molecule such as by the common phosphodiester backbone or other covalent tether of a polynucleotide molecule having a first probe segment and a second probe segment. Often, the first part and the second part of the probe reporter system are attached at either end of the common molecule of the probe, that is, at or near the 5’ and 3’ ends of the probe polynucleotide. However, alternate embodiments where one or both of the first part and a second part of the probe reporter system are tethered to an internal connection point of the common molecule are also contemplated and consistent with the disclosure herein. Systems where the first component and the second component are on distinct probes, or when one component is tethered to the analyte-binding probe or other analyte binding moiety are also contemplated herein.

[0050] In many of these cases, the probe is configured such that proximity of the first part of the probe reporter system and the second part of the probe reporter system is governed not by the probe molecule itself but by the configuration of the analyte-binding polynucleotide to which its first segment and second segment are bound. That is, the first probe segment and the second probe segment independently bind to distinct portions of an analyte-binding oligo such that proximity of the first part of the probe reporter to the second part of the probe reporter is effected by binding to the analyte-binding oligo in an analyte-binding dependent manner.

[0051] As discussed above, in some embodiments the first part and the second part of the probe reporter system are not tethered by a common molecule. In these cases, the first part of the probe reporter is tethered to a first probe segment while the second part of the probe reporter is tethered to a second probe segment which does not share a common phosphodiester backbone, and in some cases does not share any other linking covalent bond tether, or is tethered to the analyte-binding probe or other analyte binding moiety. In these cases, the first probe segment and the second probe segment are independent molecules that separately bind to distinct portions of an analyte-binding oligo, or that are brought together conditionally by biding of a first probe to the labeled analyte-binding oligo or other analyte binding moiety, such that proximity of the first part of the probe reporter to the second part of the probe reporter is nonetheless effected by binding to the analyte-binding oligo in an analyte-binding dependent manner.

[0052] Some exemplary two-part probe reports comprise, for example, fluoresceine dye (FAM and Black Hole Quencher 1 (BHQ1); DBCO-TEG (DBCO) attached via five-thymidine linker (T5) and thymine-linked Black Hole Quencher 2 (BHQ2) with cyanine 3 dye (Cy3); DBCO- TEG (DBCO) attached via five-thymidine linker (T5); thymine-linked Black Hole Quencher 1 (BHQ1); and fluoresceine dye (FAM). Alternatives include split fluorophores such as split fluorescent proteins such as split YFP or Forster resonance energy transfer(FRET)-mediated fluorophore pairs.

[0053] Alternate probes comprise single- component fluorophores or other reporters. In these systems, reporter signal is not a function of proximity of a first part of the probe reporter to a second part of the reporter. Rather, in these systems, probe binding rather than fluorophore or other reporter activity is a function of analyte-binding oligo configuration. In these systems, the probe does not bind to the analyte-binding probe unless the analyte-binding probe assumes a particular configuration, which may be either analyte bound or analyte unbound. In either case, reporter activity at a site corresponding to the analyte being assayed is a function of analytebinding probe binding to the analyte. Rather than a first part and a second part of a reporter system being held apart from one another or in proximity to one another at the site of or in proximity to an analyte-binding oligo, a signal is mediated by the ability of a probe having a single component reporter to bind to the analyte-binding oligo at its expected site. In either case, reporter signal at the site expected for an analyte signal is gated by whether the analyte-binding oligo is bound to its target analyte.

[0054] A broad range of chemistry methods may be used to covalently connect the biomolecule (nucleic acid, antibody, protein, or peptide) to a dye or other detection moiety, such as the bioconjugation methods described in Hermanson, G., Bioconjugate Techniques, Academic Press (1996), herein incorporated by reference in its entirety.

[0055] Reporter systems can comprise a dye or dye pair conjugated to the probe molecule. In some aspects, the dye pair can be a donor-acceptor fluorophore pair, in particular a FRET pair (Forster resonance energy transfer). In some aspects, the dye pair can be a fluorophore-quencher pair. In some aspects, each of the dyes can be conjugated to a separate site on the immobilized molecule.

[0056] In some embodiments, the probe comprises one or more covalently attached dyes (including a fluorescent dye). For example, the dye can be chemically linked to the probe using a cysteine or lysine residues (naturally present or available via mutations) or to the free amino group of the N-terminus.

[0057] Exemplary fluorophores include, but are not limited to, fluorescent nanocrystals; quantum dots; d-Rhodamine acceptor dyes including dichloro[Rl 10], dichloro[R6G], dichloro[TAMRA], dichloro[ROX] or the like; fluorescein donor dye including fluorescein, 6- FAM, or the like; Cyanine dyes such as Cy3B; Alexa dyes, SETA dyes, Atto dyes such as atto 647N which forms a FRET pair with Cy3B and the like. Fluorophores include, but are not limited to, MDCC (7-diethylamino-3-[([(2-maleimidyl)ethyl]amino)carbonyl]coumarin), FAM, TET, HEX, Cy3, Cy3B, TMR, ROX, Texas Red, TAMRA, Cy5, Cy7, Cy3.5, Cy7.5, LC red 705 and LC red 640. Fluorophores and methods for their use including attachment to antibodies and other molecules are described in The Molecular Probes® Handbook (Thermo Fisher, Carlsbad, California) and Fluorophores Guide (Promega, Madison, Wisconsin), which are incorporated herein by reference in their entireties. Exemplary quenchers include, but are not limited to, ZEN, IBFQ, BHQ-1, BHQ-2, DDQ-I, DDQ-11, Dabcyl, Qxl quencher, Iowa Black RQ, and IRDye QC-1. In some embodiments, the aptamer comprises a nucleotide which is a fluorophore.

[0058] In some embodiments, when the dye is a fluorophore, the fluorophore can comprise a fluorescent moiety and conjugation moiety. Exemplary fluorescent moieties can include the dyes described herein and further can be selected from : rhodols; resorufins; coumarins; xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins; phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles; stilbenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones; eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), including Cy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS and DYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY-777 and DY-782; Lucifer Yellow; CASCADE BLUE; TEXAS RED; BODIPY (boron-dipyrrom ethene) (Molecular Probes) dyes including BODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO 390, ATTO 425, ATTO 465, ATTO 610 61 IX, ATTO 610 (N-succinimidyl ester), ATTO 635 (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular Probes); DDAO (7-hydroxy-9H-(l,3-dichloro-9,9-dimethylacridin-2- one or any derivatives thereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes (LiCor) including IRDYE 700DX (NETS ester), IRDYE 80016 (NETS ester) and IRDYE 800CW (NETS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED (phosphoramidate), EPOCH YAKIMA YELLOW (phosphoramidate), EPOCH GIG HARBOR GREEN (phosphoramidate); Tokyo green (M. Kamiya, et al., 2005 Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 and CF555 (Biotium). Exemplary conjugation moieties are chemical handles which can form covalent bonds with the biomolecule and can include or exclude: NHS (N-hydroxy- succinimide), azide, tetrazine, alkyne (including strained alkyne, such as dibenzocyclooctyne group (DBCO as described in the example 3)), aldehyde, oxo-amine, imine-formation moieties (e.g., Solulink Hydrazine), maleimide, thiol, amine, and alkyl halide. In some embodiments, the fluorophore can further comprise a spacer moiety. The spacer moiety can include or exclude a polyethylene glycol polymer (for example, with 1 to 20 repeat units), polypropylene glycol polymer (for example, with 1 to 20 repeat units), polyethylene or polypropylene polymer (for example, with 1 to 20 repeat units).

[0059] In some embodiments, the probe is complexed with an intercalating dye in addition to or as an alternative to covalent labeling. The intercalating dye exhibits fluorescence when in the presence of a binding competent state which includes hybridized segments. When the environmental change induces a change in the aptamer or other affinity reagent structure such that it becomes unfolded or otherwise changes its conformation (so as to lose the persistence of the original binding competent state), the fluorescence signal decreases. The aptamer persistence loss measured with an intercalating dye can be performed in both the presence and absence of a binding partner to compare the difference, per the methods described herein. The intercalating dye can be an intercalating dye disclosed in U.S. Pat. No. 8,399,196, herein incorporated by reference. The intercalating dye can be selected from: DAPI (4',6-diamidino-2-phenylindole), 7- AAD (7-aminoactinomycin D), ethidium bromide, Hoechst 33258 (4-[6-(4-m ethyl- 1- piperazinyl)[2,6'-bi-lH-benzimidazol]-2'-yl]-phenol, trihydrochloride) (and also 33342, 34580), YOYO-l/DiYO-l/TOTO-l/DiTO-1 (YOYO-1 is also referred to as [12(2)Z,16(172)Z]-

13, 7, 7, 11, 11, 173-Hexamethyl-13H,173H-7,l l-diaza-31X5, 151X5-3(4,1), 15(1, 4)-diquinolina-

1 , 17(2)-bis([ 1 ,3]benzoxazola)heptadecaphane- 12(2), 16(172)-diene-7, 11 -diium-31,151- bis(ylium) tetraiodide), Sybr Gold ([2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3- dihydro-3-methyl-(benzo-l,3-thiazol-2-yl)-methylidene]-l-phenyl-quinolinium]), and Sybr Green (N',N'-dimethyl-N-[4-[(E)-(3-methyl-l,3-benzothiazol-2-ylidene)methyl]-l- phenylquinolin-l-ium-2-yl]-N-propylpropane-l,3-diamine), and any other DNA intercalating dye referenced in the Molecular Probes catalog identified herein.

[0060] A notable feature of many probes herein -bipartite single molecule, bipartite double molecule and signal component reporter systems - is that the probe is not designed to exhibit specificity for the analyte for which it reports. Rather, the probe is designed to bind to conserved or ‘universal’ sites, the configuration reporting moieties, that are common to a plurality, or up to in some cases most or all of the analyte-binding oligos of a system, or the probe is designed to bind specifically to a particular analyte-binding oligo rather than to its analyte.

[0061] That is, many probes are universal in that they bind analyte-binding moieties independent of the bind analyte-binding moiety identity. Alternatively, some probes additionally have a portion that is reverse-complementary to a specific region of an analyte-binding oligo or other analyte binding moiety, such that specific probe-binding moiety complexes form. This approach is particularly useful when probes rather than analyte binding moieties are bound to a surface is a position-specific manner.

[0062] As a consequence, a wide diversity of analytes may be assayed using a uniform or a single probe population. Furthermore, in some cases the probe population may be synthesized in bulk, such that aliquots drawn from a common pool may be used in multiple assays.

[0063] Surface. The analyte-binding oligos or other binders are in some cases tethered to a surface, such as a planar surface or the surface of a bead. Alternately, in some cases the analytebinding oligos or other binders are in solution, such as in a well or an emulsion droplet, or to be applied to a patterned surface. In some of these cases the probes rather than the analyte-binding oligos or other binders are tethered to a surface. When probes are tethered to the surface, the probes are often synthesized so as to have a unique or analyte-binding moiety specifying portion that is reverse complementary to a corresponding portion of the or analyte-binding moiety, such that a probe may recruit a specific analyte-binding moiety to a particular location on a surface. Alternately, analyte-binding moieties may bind to surface or bead bound probes at random. In these cases analyte binding moiety identity may be determined using decoding methods known in the art, such as those taught in Gunderson, K.L.; Kruglyak, S.; Graige, M.S.; Garcia, F.; Kermani, B.G.; Zhao, C.F.; Che, D.P.; Dickinson, T.; Wickham, E.; Bierle, J.; et al. Decoding randomly ordered DNA arrays. Genome Res. 2004, 14, 870-877, which is hereby incorporated by reference in its entirety, or in Epstein, J.R.; Ferguson, J. A.; Lee, K.H.; Walt, D.R. Combinatorial decoding: An approach for universal DNA array fabrication. J. Am. Chem. Soc. 2003, 125, 13753-13759, which is hereby incorporated by reference in its entirety. Alternate approaches for determining the identity of an analyte-biding oligo or other analyte binding moiety nonspecifically bound to a probe in solution or tethered to a surface or bead are known in the art and consistent with the disclosure herein.

[0064] Substrates are generally locally flat. Some substrates are flat surfaces, such as those of a chip or a region of a flow cell. Alternately, some substrates are locally flat beads, such as spherical beads. Many of these beads comprise one cluster per bead, the cluster being homogeneous or heterogeneous. Beads may be deposited in wells, such as wells that are configured to accommodate no more than one bead per well. Beads in these cases are often configured to be amenable to spatial decoding or to sequencing reactions, such that a bead in a well may be amendable to detection of its binding moiety and therefore the presence of its target analyte at a position on a well array or other surface or liquid array.

[0065] The decoding may comprise sequencing of a barcode or other distinct tag identifier. Alternately, particularly in the case of aptamers, the aptamers of a cluster are sequenced directly for a bead in a well or a position on a surface or of a liquid array so that the signal from that source may be correlated to detection of a particular target analyte.

[0066] Alternatively, the identity of each bead on the array can be determined by a decoding process. In one embodiment, the decoding process identifies the different bead types by using sequential hybridization of pools of fluorescently-labeled complementary decoder probe sequences (Gunderson et al. 2004, above; Vickovic et al. “High-definition spatial transcriptomics for in situ tissue profiling” Nat Methods. 2019 Oct;16(10):987-990). The decoder probes are stripped from the bead array between decoder pool hybridization steps. [0067] Alternately, in some cases affinity reagents such as aptamers or antibodies are deposited into an aqueous volume such as a well or an emulsion droplet without being bound to a solid surface.

[0068] Analyte-bound surfaces are subjected to a range or gradient of environmental condition change, such that as the melting temperature (Tm), melting voltage (Vm) or other threshold parameter for a given environmental condition is met or passed, or when the level of persistence is surpassed for a given analyte-binding moiety pair, a corresponding change in fluorescence is observed. When the threshold parameter is consistent with signal persistence caused by target analyte binding, the sample may be scored as having the target analyte or analytes.

[0069] Alternately, in some cases a single environmental condition value is selected, such as a single voltage or a single temperature, which is known to fall outside of the stability range for an unbound binding moiety such as an aptamer, but to fall within the persistence stability range for that binding moiety bound to a target analyte. The surface is subjected to that temperature (or other environmental perturbation) and binding moiety fluorescence is assayed. This approach may be used for a single temperature or other environmental perturbation, or may comprise a plurality of temperatures or other environmental perturbations selected to visualize secondary structure persistence for a plurality of binding moieties having various different Tm, Vm or other threshold parameters. In some cases, a single environmental condition value is selected, and a plurality of samples are run across or contacted to the surface successively such as over time, so as to effect iterative sampling or assaying of samples such as temporally distinctly collected or provided samples.

[0070] Consistent with single environmental perturbation assays, in some cases the binding moieties for a given surface are selected so as to have Tm, Vm or other threshold parameters, or persistent shifts that all fall within a common range, such that a single temperature or other environmental shift is sufficient to visualize the target analyte-sensitive fluorescence status for a substantial proportion or all of the binding moiety clusters on a surface. That is, in some cases a single application of a temperature is sufficient to assay for target analyte binding status for a plurality of clusters, or such that a surface may be assayed through a single temperature or other environmental perturbation, rather than subjecting a surface to a temperature or other environmental perturbation gradient so as to span a plurality of Tm, Vm, or other threshold parameter thresholds or to fall within a plurality of persistence ranges.

[0071] In some embodiments, a “cluster” (a clonal population of immobilized molecules all having the same identity and immobilized within a single spot) may be attached to a support surface such as an essentially planar substrate, bead, microparticle, or nanoparticle as contemplated herein.

[0072] The size of each cluster can range from about 30 microns in diameter, and have a circular or semicircular shape. In some embodiments, there can be up to 2000 aptamer clusters arrayed on a 2 mm x 2 mm CMOS sensor substrate, which will allow for a point-of-care device that simultaneously, inexpensively, and rapidly detects ~60 analytes of choice present in different biological solutions. However, a broad range of shapes and cluster sizes are consistent with the disclosure herein. In some cases cluster size is limited by the optical capacity of the detection device, such that cluster sizes are limited by the pixel size of the detection device. Alternately, larger clusters are also consistent with some embodiments of the technology, such as 50, 100, 200, 300, 400, 500 or greater than 500 microns. Some surfaces are 'coated' with constituents of a single cluster, such that all or a substantial portion of the surface comprises a single binding moiety lawn rather than distinct clusters.

[0073] Accordingly, disclosed herein are surfaces for detection of a target analyte, such as surfaces comprising one or more of the following elements: a plurality of aptamer clusters, wherein a first cluster of the plurality of clusters comprises a first aptamer having a first configuration, wherein the aptamer first configuration is sensitive to presence of a first analyte; and wherein a second cluster of the plurality of clusters comprises a second aptamer having a second configuration, wherein the aptamer second configuration is sensitive to presence of a second analyte.

[0074] Similarly disclosed herein are surfaces for detection of a target analyte, such as surfaces comprising one or more of the following elements: a plurality of probe clusters, wherein a first cluster of the plurality of clusters comprises a first probe having a first analyte-binding moiety specifying segment so as to specify a first analyte binding aptamer having a first configuration, wherein the aptamer first configuration is sensitive to presence of a first analyte; and wherein a second cluster of the plurality of clusters comprises a second probe having a second analytebinding moiety specifying segment so as to specify a second analyte binding aptamer having a second configuration, wherein the aptamer second configuration is sensitive to presence of a second analyte. [0075] Some aptamer bound surfaces comprise a third cluster of the plurality of clusters comprises an aptamer having a first configuration, wherein the aptamer first configuration is sensitive to presence of a first analyte.

[0076] Some surfaces comprise a third cluster of the plurality of clusters comprises a chimeric aptamer comprising at least a binding moiety of the first aptamer and at least a binding moiety of the second aptamer.

[0077] In some cases at least some of the plurality of clusters are homogenous as to aptamer composition. Alternately, in some cases at least some of the plurality of clusters are heterogeneous as to aptamer composition. Often, at least one of the plurality of clusters consists of the first aptamer, while at least one of the plurality of clusters consists of the second aptamer. [0078] At least some of the plurality of clusters comprise a single affinity reagent such as an aptamer population per cluster in some cases. An aptamer of the aptamer clusters often comprises a detection moiety such as a fluorophore, and in some cases also comprises a quencher, or comprises a fluorophore acceptor pair.

[0079] Some surfaces are configured such that individual affinity reagents such as aptamers of a set of clusters of the plurality of clusters bind to a set of analytes implicated in a common biological process, such as a signaling pathway, for example a cancer pathway, a cancer progression evaluation pathway, a disease response pathway, a pathogen cell cycle pathway, or other disease related pathway.

[0080] Binding the first analyte or a first analyte-analyte binding moiety complex to the surface often comprises delivering the analyte in an aqueous solution. Sometimes, binding the first analyte to the surface does not require processing the analyte from a sample. Alternately, some samples are processed prior to contacting to the surface, for example by enrichment, extraction, or buffering. In some cases a surface is washed subsequent to sample binding, but prior to assaying for target analyte presence. Exemplary washes include buffers, such as PBS, PBST, TBS, TBST, or others.

[0081] In various surfaces herein, the plurality of aptamer clusters or probe clusters comprises at least 100 clusters, such as 100, 200, 300, 400, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000 or more than 200,000 clusters. A number of cluster sizes are consistent with the disclosure herein, for example a diameter of at least about 10 microns, 20 micros, 30 microns, 50 microns, 100 microns, 200 microns or 500 microns, among others. The plurality of aptamer clusters may exhibit a range of cluster pitches, for example at least about 20 um, 40um, 60um, 80um, lOOum, 200um, 500um or greater. The plurality of aptamer clusters exhibit a broad range of affinity reagent densities such as aptamer densities, such as about 10el4 aptamer molecules per cm2, or even 10el3, 10el2, lOel l, lOelO, 10e9, 10e8, 10e7 or less than 10e7. In some selected surface configurations, a plurality of affinity clusters such as aptamer clusters each exhibit an analyte bound conformational change at about the same temperature, such as within 0.1, 0.2, 0.5, 1, 2, 3, 4, or 5 degrees Celsius.

[0082] Similarly disclosed herein are systems for analyte detection. Some such systems comprise some or all of the following elements: a surface comprising a plurality of aptamer clusters; a surface condition modulator; and an imaging apparatus. Some such systems comprise some or all of the following elements: a surface comprising a plurality of probe clusters; a surface condition modulator; and an imaging apparatus.

[0083] The system in some cases does not comprise moving parts. Alternately or in combination, some systems do not comprise a microfluidics pump or do not comprise fluid piping. The surface is in some cases an interior of a flow cell. The affinity reagent such as aptamer clusters are present at a cluster pitch of about 40um, or in some cases at least about 10 um, 20 um, 40 um, 60 um, 80 um, 100 um, 200 um, 500 um or greater. The affinity clusters such as aptamer clusters may comprise aptamers of a common cluster that bind a common target. [0084] Generally, the clusters or the analyte-binding oligos tethered to the surface or contained in emulsions need not be labeled. Labels are instead delivered through the labeled probes, which do not exhibit analyte specificity and can be manufactured in bulk..

[0085] The condition modulator of the systems herein regulates a conformational disruptor condition, such as temperature, ion concentration, electric field, current, voltage, vibration, sonication intensity, magnetic field or other disruptive parameter mentioned herein. In many embodiments the condition is temperature. Alternately or in combination, the condition modulator regulates a buffer condition.

[0086] A number of imaging apparatuses are consistent with the disclosure herein, such as a digital camera or a digital phone. Some imaging apparatuses are provided with the system herein, while others are independent. In some cases the imaging apparatus is fixed to the surface. [0087] The plurality of affinity reagent such as aptamers variously comprises at least 1,000 clusters having distinct aptamer, alternately 2,000, 5,000, 10,000, 20,000, 50,000 or more than 50,000. Similarly, the plurality of aptamer clusters comprises aptamers targeting at least 1,000 distinct target analytes, alternately 2,000, 5,000, 10,000, 20,000, 50,000 or more than 50,000. [0088] Alternately, the plurality of aptamer clusters comprises aptamers targeting at least 10 distinct target analytes, or even 5, 4, 3, 2 or a single target analyte.

[0089] The assaying is completed in no more than 5 minutes, or no more than 5, 6, 7, 8, 9, 10, 15, 20, or 30 minutes, in particular when the change in the destabilizing condition is effected gradually or incrementally, such as through subjecting the sample to a gradient. Other durations are also consistent with the disclosure herein. [0090] Alternately, the assaying is completed in no more than 30 seconds, such as when the assaying comprises assaying at a single destabilizing condition or changing from a first to a second and optionally to a third condition parameter. Exemplary times are no more than 10, 15, 20 30 45 or 60 seconds, or no more than 1, 2, 3, 4, or 5 minutes. Other durations are also consistent with the disclosure herein.

[0091] Assaying exhibits very high sensitivity in some embodiments, such that in some cases assaying is sensitive to an analyte at a concentration of at least 1 fM, or alternately at least lOfM, lOOfM, IpM, lOpM, lOOpM, InM, lOnM, lOOnM, luM or greater than 1 uM.

[0092] A number of sample types are consistent with the disclosure herein, such as a body fluid such as blood, for example in a droplet of at least luL, 2uL, 5uL, lOuL, 20uL, 50uL, or greater than 50uL. Alternate body fluids, such as plasma, saliva, sweat, bile, urine or other fluids are similarly consistent with the disclosure herein, such as in the volumes listed.

[0093] In some selected surface configurations, a plurality of affinity clusters such as aptamer clusters each exhibit an analyte bound conformational change at about the same temperature, such as within 0.1, 0.2, 0.5, 1, 2, 3, 4, or 5 degrees Celsius. This facilitates rapid assays in some cases, as the shift to a single assay temperature or a narrow assay temperature range is sufficient to assay for a broad range of target analytes having a similar temperature at conformational change.

[0094] Also disclosed herein are methods of assaying for an analyte in a sample, comprising one or more of the following elements: contacting the sample to a surface comprising a plurality of aptamer populations, changing a condition at the surface, and assaying for a change in at least one analyte configuration.

[0095] The assaying is completed in no more than 5 minutes, or no more than 5, 6, 7, 8, 9, 10, 15, 20, or 30 minutes, in particular when the change in the destabilizing condition is effected gradually or incrementally, such as through subjecting the sample to a gradient. Other durations are also consistent with the disclosure herein.

[0096] Alternately, the assaying is completed in no more than 30 seconds, such as when the assaying comprises assaying at a single destabilizing condition or changing from a first to a second and optionally to a third condition parameter. Exemplary times are no more than 10, 15, 20 30 45 or 60 seconds, or no more than 1, 2, 3, 4, or 5 minutes. Other durations are also consistent with the disclosure herein. Often, these low assaying times are enabled by selecting affinity reagents such as aptamers having common temperature shifts for their target analyte binding. [0097] Assaying exhibits very high sensitivity in some embodiments, such that in some cases assaying is sensitive to an analyte at a concentration of at least 1 fM, or alternately at least lOfM, lOOfM, IpM, lOpM, lOOpM, InM, lOnM, lOOnM, luM or greater than 1 uM.

[0098] A number of sample types are consistent with the disclosure herein, such as a body fluid such as blood, for example in a droplet of at least luL, 2uL, 5uL, lOuL, 20uL, 50uL, or greater than 50uL. Alternate body fluids, such as plasma, saliva, sweat, bile, urine or other fluids are similarly consistent with the disclosure herein, such as in the volumes listed.

[0099] In some selected surface configurations, a plurality of affinity clusters such as aptamer clusters each exhibit an analyte bound conformational change at about the same temperature, such as within 0.1, 0.2, 0.5, 1, 2, 3, 4, or 5 degrees Celsius. This facilitates rapid assays in some cases, as the shift to a single assay temperature or a narrow assay temperature range is sufficient to assay for a broad range of target analytes having a similar temperature at conformational change.

[0100] In some cases, the analyte comprises a protein. The method may variously distinguishes the protein according to a post-translational state of the protein, such as phosphorylation state of the protein or a glycosylation state of the protein, such as a hemoglobin glycosylation state (e.g. HbAlc).

[0101] The analyte, or an analyte of the sample, may alternately or in combination comprise a small molecule, metabolite, carbohydrate, a nucleic acid, a lipid, an epitope, a cell or cellular component, a virus or a virus component, or other analyte as listed herein.

[0102] Disclosed herein are methods of assaying for an analyte in a sample, comprising one or more of the elements of contacting the sample to a surface comprising a plurality of affinity reagents such as aptamer populations, assaying for affinity reagents such as aptamer population first signal such as fluorescence, changing a condition at the surface, and assaying for affinity reagents such as aptamer population second signal such as fluorescence.

[0103] The changing of a condition in some cases comprises a gradual changing, and similarly, assaying for aptamer population second fluorescence comprises gradual assaying. In some cases changing a condition comprises a continuous changing, while in alternate cases changing a condition comprises a discrete changing.

[0104] The surface comprises wells in some cases and the aptamer populations are segregated into wells. Similarly, in some cases the surface comprises wells, and the aptamer populations are immobilized on beads, and the beads are localized into the wells. Often, the wells accommodate no more than one bead per well. To identify a bead in a particular well, the methods comprise sequencing a tag associated with a bead in a well, or sequencing an aptamer associated with a bead in a well, or both sequencing an aptamer and a tag. [0105] Also disclosed herein are methods of distinguishing among analytes in a sample, comprising one or more of the elements of binding an unknown analyte to an affinity reagent such as an aptamer that binds a first analyte and a second analyte, changing a condition at the analyte and concurrently measuring fluorescence at the analyte, and observing a change in an output such as fluorescence, wherein the first analyte causes a change in aptamer fluorescence at a first change in the condition, and the second analyte causes a change in aptamer fluorescence at a second change in the condition. The aptamer is often tethered to a surface, such as a surface is covered with a liquid at the aptamer.

[0106] Arrays. This disclosure provides for biosensors, in particular biosensors for detecting the presence of an analyte in a sample according to the methods described herein. The biosensor can comprise a plurality of immobilized molecule clusters (e.g., immobilized biomolecule) configured on a surface as an array. The array is an affinity reagent array, such as an antibody array or an aptamer array. In some embodiments, the array can be made by contacting aptamers functionalized as described above with a functionalized surface using an "inkjet" like technology, wherein a microdroplet (or smaller volume than a microliter) is deposited at a selected location on the substrate. In some embodiments, the functionalized substrate can be prepared by treating with oxygen plasma followed by water for surface activation and silanol repopulation. In some embodiments, the functionalized substrate can be prepared without water treatment. The functionalized substrate can be prepared using a commercial CVD/plasma system (e.g., EasyTube 100, FirstNano (NY)). The functionalized substrate can be functionalized with an appropriate bioconjugation target appropriate for the bioconjugation site partner on the functionalized aptamer. For example, after aptamer sequence identification, the oligonucleotide sequence can be commercially sourced (e.g., IDT DNA) with a 5'-amino modifier. The surface can be functionalized with a NHS (N-hydroxy succinimide) functionalized silane (Gelest). The aptamer can be contacted with the NHS-functionalized surface to react the 5'-amino modified aptamer to the NHS-moiety on the surface to covalently bond the aptamer to the surface. Numerous methods exist to conjugate oligonucleotides to surfaces which can include or exclude thiol/maleimide, click chemistry (azide/alkynyl, e.g., dibenzocyclooctyne (DBCO)), carboxylic acid/amine, carboxylic acid/alcohol, amine/halide, etc., using appropriate bioconjugation pairs. [0107] In some embodiments, the immobilized molecules, or clusters of the same immobilized molecule identity, can be isolated from immobilized molecules of a different identity, and all immobilized molecules subject to an environmental perturbation.

[0108] The immobilized molecules are variously analyte-binding oligos or other analyte binding moieties, or are probes, such as probes having a at least single region reverse complementary to an analyte binder or having at least two regions reverse complementary to an analyte binder. Probes may in some cases comprise a specificity domain that is reverse complementary to a particular analyte-binding moiety. Alternately, some arrays comprise probes that are not specific to any particular analyte binding moiety. In these cases, analyte-binding moiety and in some cases the bound analyte at a probe site is identified using a decoding approach known in the art, such as that of Gunderson 2004 or other references provided elsewhere herein.

[0109] After deposition of the aptamer solution onto the functionalized substrate, the aptamer cluster can be of a number of shapes, such as round and have a diameter from 1 to 500 microns, based on the concentration, spotting solution composition, temperature, humidity, and surface density of functional molecules. In some embodiments, the aptamer cluster is about 30 microns in diameter with a pitch of about 45 microns. The pitch can range between 10 microns to several millimeters. The pitch can be controlled by the selective deposition locations. The purpose of decreasing the pitch is to increase the number of aptamer clusters per unit area. However, the pitch must not be too short to reduce the likelihood of crossover from one aptamer cluster to a neighboring aptamer cluster. With the pitch at 45 microns and the cluster diameter at 30 microns, the resolution (or density per unit area) of the aptamer clusters will be about 600 dpi. In some embodiments, clusters can be formed on selectively functionalized substrates, so as to enforce an ordered array of aptamer clusters. Aptamer clusters will therefore be comprised of tens of millions of individual aptamers and their fluorescent signal will be easily resolved above detection limit with modified commercial CMOS sensors. Each aptamer cluster can be printed in replicate (e.g., from 2 to 100 replicates or more, preferably 3 to 50, and more preferably 4) to ensure at least three independent measurements for each aptamer type in case one of the clusters fails to perform during the test or manufacturing process, and/or to obtain statistical reproducibility and error measurement associated with the assay. With a typical 45 micron pitch, about 2,000 clusters can be fit on a 2mm x 2mm substrate. Such a configuration affords the simultaneous detection of about 60 different analytes in a biological sample which should be more than sufficient for most clinical applications because typical blood test panels analyze between 5 and 30 analytes.

[0110] Alternately, arrays are in some cases formed through the generation of a population of cluster coated beads, which may be assayed individually, in a linear series through microfluidics, or for example may be deposited onto a surface, such as into wells on a surface. The clusters, again, may comprise analyte binding moieties or specific or nonspecific probes. Reporters or reporter pairs may be bound to the probe or bound to both the probe and the analyte binding moiety, as contemplated in the description of probes elsewhere herein.

[OHl] The wells on such a surface are often configured to accommodate no more than one bead at a time, such that well positions effectively act as cluster positions in these systems. To determine the identity of the bead cluster deposited within a particular well, a signal identifying the bead is generated. Such a signal may comprise sequencing a tag or label tethered to the beads, or may comprise sequencing aptamers of an aptamer cluster of the bead directly, thereby identifying the bead and the affinity reagent responsible for the analyte signal at that position or that well in a well array.

[0112] Alternatively, the identity of each bead on the array can be determined by a decoding process. In one embodiment, the decoding process identifies the different bead types by using sequential hybridization of pools of fluorescently-labeled complementary decoder probe sequences (Gunderson et al. 2004; Vickovic et al. 2019). The decoder probes are stripped from the bead array between decoder pool hybridization steps.

[0113] In some cases beads beneficially comprise an oligo tag per bead, such that sequencing the oligo tag identifies the target analyte bound by the bead. Alternately or in combination, sequencing the aptamer tethered to the bead in the well identifies the target analyte bound to the bead at the position. Alternately, the tag is decoded using methods described herein or known in the art.

[0114] In yet further alternatives, affinity reagents are deposited as beads or unbound into isolated volumes, such as emulsions or wells of an array. Aliquots of a sample are deposited into the isolated volumes, and binding assays are performed as disclosed herein. The affinity binding reagent is in some cases known prior to deposition at a particular isolated volume such as a well position. Alternately, in some cases an affinity reagent is isolated subsequent to target analyte assaying, and is then its identity is determined through sequencing of the affinity reagent aptamer directly, or of a tag co-deposited with the affinity reagent in the isolated volume.

[0115] The arrays disclosed herein are compatible with harboring affinity reagent populations such as those disclosed above, and for use in the systems and methods disclosed throughout the present disclosure.

[0116] A key feature of many of the surface or array embodiments disclosed herein is that the analyte-binding oligos or other binders are deposited such that their individual locations are known or can be readily determined, for example using barcodes or other identifying oligos. Accordingly, when a signal indicative of a target analyte presence or absence is detected from an array such as a surface array, one may determine the identity of the analyte from the position of the signal on the surface. Notably, detection is not dependent upon each analyte-binding oligo or other binder having a unique or distinctive melting temperature or other binding dissociation property.

[0117] Consequently, the number of analytes to be assayed by a particular surface is not limited by the need for unique or distinctive biochemical properties of the interactions between analyte- binding oligos or other binders and their target analytes. Rather, the number of analytes to be detected is limited by the available surface area of the surface given chosen cluster size, by optical or other visualization constraints, or by other factors independent of interactions between the analyte-binding oligos or other binders and their target analytes.

[0118] Accordingly, surfaces or other detection arrays may be configured having, for example, at least 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000 or more analyte-binding oligos or other binders or clusters of analyte-binding oligos or other binders. These analytes may be assayed in a single reaction or assay round, using a single population of individual or bipartite probes. The analyte-binding oligos or other binders or clusters of analyte-binding oligos or other binders may each be unique relative to other clusters or individual binders, or may comprise duplicates, such that the same analyte is assayed at distinct clusters. In some cases distinct clusters comprising distinct analyte-binding oligos or other binders or distinct or uniform probes may nonetheless bind a common target analyte or common analyte binding moiety under identical or nonidentical parameters such as melting temperature or minimum concentration at which the analyte is bound.

[0119] Flow cells In some embodiments, the detection disclosed herein is facilitated by the use of a reaction vessel. A "reaction vessel" is a substrate to which a molecule can be immobilized or otherwise localized. The reaction vessel allows for the presentation of an applied environmental perturbation to the immobilized molecule in (or on) the reaction vessel. In some embodiments, the reaction vessel is a flow cell or chamber, multi-well plate, bead, emulsion of beads or other volume capable of hosting a plurality of clusters. Flowing liquid reagents (which can include or exclude one or more of the sample, an analyte, probe populations or analyte binding oligo or other analyte binder populations or a wash solution) through the flow cell, which contains an interior solid support surface (e.g., a surface) conveniently permits reagent exchange or replacement. Immobilized to the interior surface of the flow cell is one or more immobilized molecules using the methods described herein. In some embodiments, flow cells are in fluidic communication with microfluidic valving that permits delivery of liquid reagents (e.g., components of the "reaction mixtures" discussed herein) to an entry port. Liquid reagents can be removed from the flow cell by exiting through an exit port. Optionally, liquid reagents can be moved back and forth within the flow cell, for example, to effect mixing.

[0120] CMOS Substrates. In some embodiments, a biosensor compatible with the disclosure herein comprises: (a) an excitation light source; (b) an array of detection elements comprising a plurality of sets of stacked layers, comprising: (i) a first set of stacked layers comprising a top layer which is a transparent conductive layer configured to support an immobilized molecules; and (ii) a second set of stacked layers comprising an optical filter (e.g. interference filter) and a solid-state photodiode array; wherein the excitation light source can be coherent or incoherent, wherein the light excitation source may include an optical collimator, wherein the photodiode array comprises an array of a photoelectric transducer unit which converts the photons of received light to electrons, wherein the photoelectric transducer unit includes an active area and an inactive area, wherein the number of electrons generated by a single photoelectric transducer is proportional to the number of received photons in the active area, wherein the inactive area includes electronic and digital circuitry needed to operate the photoelectric transducer unit, wherein the transparent conductive layer is acting as a resistive heater and the voltage source circuitry apply controlled voltage across the transparent conductive electrode, with a hot contact and a ground contact, wherein the optical filter is operably coupled to the transparent conductive layer and the solid-state photodiode array, wherein a passivation layer (e.g. SiO2) may be present between the transparent conductive layer and the optical filter, wherein the immobilized molecules comprises a nucleic acid sequence, a dye pair, and one or a plurality of binding competent state(s), wherein the optical filter comprises a plurality of vertical alternating dielectric layers (e.g. interference filter) which are laterally separated by a dielectric grid with an optically opaque surface (GRO), wherein a unit cell of the GRO defines the lateral boundaries of a detection element (i.e. pixel), wherein the GRO is laid over the inactive of the photoelectric transducer array, wherein the GRO comprises a stack of dielectric and metal oxide layers in which conductive routing layers may be embedded for electric connectivity, wherein the height and the pitch of the GRO define the field of view of the photodiode array (FOV), wherein the dye pair is configured to be positioned within the FOV and the excitation light source is configured to be exterior to the FOV, and wherein the filter layers are configured to transmit the emission signal from the dye pair when the dye pair is subject to a light source from the excitation light source, and to reflect the background excitation light that is not blocked by the walls. The operable coupling between the optical filter and the transparent conductive layer can be a physical connection. In some embodiments, the dye pair of the biosensor can be covalently linked to the nucleic acid sequence. In some embodiments, the solid-state photodiode array can be fabricated using CMOS technology. In some embodiments, the solid-state photodiode array can be configured to detect the emission signal from the dye pair when the dye pair is subject to a light source from the excitation light source.

[0121] In some embodiments, the biosensor comprises a detector surface that can be functionalized (e.g. chemically or physically modified in a suitable manner for attaching an immobilized molecule). For example, the detector surface can be functionalized and can include a plurality of reaction sites having one or more biomolecules immobilized thereto. The detector surface can have a reaction array of reaction recesses. Each of the reaction recesses can include one or more of the reaction sites. The reaction recesses can be defined by, for example, an indent or change in depth along the detector surface. In other examples, the detector surface can be planar.

[0122] In some embodiments, the biosensor comprises a CMOS photodetector array. The CMOS photodetector array comprises a sensor array as described herein. In some embodiments, the CMOS photodetector array can include a plurality of stacked conductive routing layers (e.g. conductors, traces, vias, interconnects, etc.) that are capable of conducting electrical current, such as the transmission of data signals that are based on detected photons. A photodetector array comprises an integrated circuit having a planar array of the light sensors (i.e. photoelectric transducers). The circuitry formed within detector can be configured for at least one of read out signals from light sensors after an exposure period (integration period) in which charge accumulates on light sensor, signal amplification, digitization, storage, and processing. The circuitry can collect and analyze the detected emissions signal light and generate data signals for communicating detection data to a bioassay system. The circuitry can also perform additional analog and/or digital signal processing in detector. Light sensors can be electrically coupled to circuitry through gates.

[0123] In some embodiments, the solid-state photodetector array can comprise a detector which can be provided by a solid-state integrated circuit detector such as a CMOS integrated circuit detector or a CCD integrated circuit detector. The detector according to one example can be an integrated circuit chip manufactured using integrated circuit manufacturing processes such as complementary metal oxide semiconductor (CMOS) fabrication processes.

[0124] The resolution of the biosensor array is defined as the number of pixels allocated for each reaction sight, which can be as small as 1 pixel per reaction sight, or can be greater than about 50 megapixels per reaction sight.

[0125] The detector can include a plurality of stacked layers including a sensor layer, which can be a silicon layer. The stacked layers can include a plurality of dielectric layers. In the illustrated example, each of the dielectric layers includes metallic elements (e.g. W (tungsten), Cu (copper), or Al (aluminum)) and dielectric material, e.g. A12O3, Si3N4, SiO2. Various metallic elements and dielectric material can be used, such as those suitable for integrated circuit manufacturing. However, in other examples, one or more of the dielectric layers can include only dielectric material, such as one or more layers of SiO2.

[0126] In some embodiments, the field of view is from about 0.25 micron square to about 2.5 cm2. In some embodiments, the field of view can be from about 100 micron square to about 1000 mm2. In some embodiments, the field of view is 5 microns by 5 microns. In some embodiments, the field of view is 100 mm by 100 mm. In some embodiments, the field of view is round. In some embodiments, the field of view is square-shaped.

[0127] Analytes. A broad range of analytes may be detected using the disclosure herein. Exemplary analytes include small molecules, hormones, proteins, nucleic acids, carbohydrates, cells or cellular structures, virus particles or virus constituents. Presence of one analyte or type of analyte is not mutually exclusive with many other analytes, such that one may concurrently detect a broad range of analytes concurrently. In some embodiments, the analytes comprise at least some non-nucleic acid targets, or a majority of non-nucleic acid targets, or the target analytes do not comprise nucleic acids. The analytes may be similar biochemically such that they are readily enriched together. Alternately, or in combination, some of the analytes may be involved in a common signaling or other biochemical pathway.

[0128] Analytes are detected across a broad range of concentrations. In some cases, analytes are detected at a concentration of as low as or less than IfM, lOfM, lOOfM, IpM, lOpM, lOOpM, InM, lOnM, lOOnM or greater.

[0129] In some cases similar analytes may be independently identified, or quantified, through the disclosure herein. For example, proteins may be distinguished from one another by post- translational modification, such as phosphorylation status, glycosylation status, alkylation, lipidation, myristylation, carbonylation, glycosylation, or other post-translational modifications known in the art or otherwise identified. Similarly, nucleic acid modifications may be distinguished from unmodified variants, such as methylation, pseudouridylation, 2-O- methylation or other modification.

[0130] As an example, methods and systems herein may detect, distinguish and quantify the difference between glycosylated and total hemoglobin proteins in a blood sample (e.g., HbAlc test), phosphorylated or unphosphorylated RPS6 protein, phosphorylated or unphosphorylated cell cycle proteins such as p53, nucleosome or histone acetylation status, protein ubiquitination status, or other modifications.

[0131] Alternately, post-translational modifications or allelic variants in proteins may be identified de novo through the disclosure herein, for example by observing a shift in a target analyte binding affinity to a affinity reagent cluster relative to an expected binding affinity. That is, identification of a target analyte at a cluster that comprises aptamers that bind that target at a known binding affinity, but observing that target analyte to have a binding affinity that differs from an expected binding affinity, may indicate that the target analyte harbors a post- translational modification or an allelic variation, in the case of proteins for example, that slightly impacts without abolishing binding affinity. The shift in binding affinity can manifest itself in a shift of temperature at which fluorescence is abolished of, for example, at least, at most or about 1, 2, 3, 4,5, 6, 7, 8, 9,10 or more than 10 degrees Celsius, or a shift in temperature or other environmental condition or disruptive force magnitude of, for example at least, at most or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or greater.

[0132] The analyte can be a small molecule, protein, carbohydrate, peptide, antigen, polymer, and the like. In some embodiments, the analyte can be a species of a Comprehensive Metabolic Panel. A typical Comprehensive Metabolic Panel (CMP) can include or exclude any of the following analytes, which can be detected using the methods described herein: Glucose, Calcium, Sodium, Potassium, Bicarbonate (an electrolyte that reflects the level of carbon dioxide (CO2) in the sample), Chloride, Blood urea nitrogen (BUN), Creatinine, Albumin, Total protein (the sum of albumin and globulins), Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and Bilirubin. Often, target analytes comprise at least some non-nucleic acid targets, or a majority of non-nucleic acid targets.

[0133] In the broadest embodiments, the disclosure herein is consistent with detection of any analyte for which an affinity reagent binding partner can be identified.

[0134] Some target analytes are subjected to repeated, iterative or ongoing detection, such as through the repeated flow of samples across the surface of an array comprising affinity reagents configured to assay for target analyte presence. An example of such a configuration is a glucose sensor, such as one that iteratively receives blood samples and assays for glucose levels in the samples.

[0135] Correlating signals to analyte locations. Consistent with the compositions and systems herein, disclosed are methods for high-throughput analysis a sample for the presence or abundance of one or a plurality of analytes. Some such methods comprise administering at least one sample composition to an array of analyte-binding oligos or other binders, such that an interaction between an analyte-binding oligo or other binder and its target analyte results in a change in configuration of its configuration-reporting moieties, or common bipartite universal detection oligo binding sites, such that the configuration of the configuration-reporting moieties, or common bipartite universal detection oligo binding sites is indicative of analyte binding status. Reporter moieties such as a fluorophore quencher pair are located on a probe or alternately are distributed between the probe and the analyte binding moiety, such that interactions between the analyte and the analyte binding oligo or other analyte binding moiety mediate the colocalization of reporter moiety pairs so as to mediate reporter activity.

[0136] Concurrently with, prior to or subsequent to sample administration, the array is contacted to a uniform composition comprising labeled probes having a first segment and in some cases a second segment that independently bind to one or more parts of the configuration-reporting moieties, or common bipartite universal detection oligo binding sites. In some cases binding is between two parts of common bipartite universal detection oligo binding sites, as in Fig., 1, while alternately a single common universal detection oligo binding site may be used, as in Fig. 2.

[0137] A broad range of analytes may be assayed for in a sample, such as proteins, carbohydrates, oligos or other nucleic acids, lipids, or other cellular or noncellular components. In some cases the list of analytes to be assayed does not comprise oligos or other nucleic acids. In some cases distinct isomers or post-translational variants of a protein may be differentially assayed for and their relative or absolute proportions quantified, such as that of phosphorylation variants, methyl- or acetyl- variants, selenocysteine residues or other post-translational variant. [0138] The labeled probes in some case comprise a first part and a second part of a probe reporter system, and are configured such that proximity of the first part of the probe reporter system and the second part of the probe reporter system is governed not by the probe molecule itself but by the configuration of the analyte-binding polynucleotide to which its first segment and second segment are bound. The first part and a second part of a probe of the probe system are alternately tethered to one another or are independent molecules. In the latter case, a uniform population of probes comprises both the first independent part and a second independent part of a bipartite probe, as shown in Fig. 1.

[0139] Alternately in some cases the probe is configured such that binding to each part of the configuration-reporting moieties, or common bipartite universal detection oligo binding sites, is dependent upon analyte binding probe configuration.

[0140] In yet additional alternatives, the labeled probes comprise a first part of a probe reporter system, and are configured such that proximity of the first part of the probe reporter system and a second part of the probe reporter system on an analyte binding moiety is governed not by the probe molecule itself but by the configuration of the analyte-binding moiety to which its first segment is bound, as shown inf Fig. 2.

[0141] Notably, the probes used herein are not designed for specificity to any particular analyte. Rather, the probes are designed to anneal to the configuration-reporting moieties, or common single part or bipartite universal detection oligo binding sites of the analyte-binding oligos or other binders. Accordingly, only a single uniform population of single molecule probes or bipartite probes needs to be synthesized pursuant to some of the methods herein. This dramatically reduces assay cost and increases assay throughput.

[0142] Alternately, some probe populations are synthesized to show affinity or specificity to one or a subset of analyte binding moieties, such as through a reverse-complementary region that targets a particular analyte binder or analyte binders. [0143] Common to both of these approaches, the probes used herein are not designed for specificity to any particular analyte.

[0144] In exemplary embodiments the array comprises a surface onto which unlabeled analytebinding oligos or other binders are located at either known locations or locations tagged, for example with assayable tags such s barcodes. Alternately, arrays comprise emulsion droplets or wells on a surface into which barcoded beads harboring unlabeled analyte-binding oligos or other binders are deposited.

[0145] A common feature of many of these arrays is that the analyte-binding oligos or other binders do not harbor fluorophores or other reporters. That is, individual analyte-binding oligos or other binders do not need to be labeled using fluorophores or other reporters, which dramatically reduces the cost and complexity of array synthesis.

[0146] In alternate embodiments, split reporters are shared between a probe and an analytebinding oligo or other analyte binder. A portion of the reporter is added to the analyte-binding oligo or other analyte binding moiety, often at the 5’ end of an oligonucleotide sequence, and may be added through a primer extension reaction pursuant to analyte binder synthesis. Deploying half of a split reporter system onto the analyte binder allow flexibility in reporter configurations, which is particularly useful in cases where reporter position may impact analytebinding moiety binding configuration. An example of this configuration is provided in Fig. 2. [0147] Subsequent to or concurrently with sample contacting to the array, reporter activity is assayed for across the array. Reporter activity may also be assayed for prior to probe binding. Reporter activity is assayed for once or multiple times, such as under multiple conditions. In some cases reporter activity is assayed for continuously or at multiple time points as the array is subjected to a temperature gradient or interval temperature incubations across a temperature range. Alternately or in combination, arrays are assayed while at distinct environmental conditions, such as temperatures, for example a low temperature corresponding to analytebinding oligos or other binders in a ‘tight’ or closed configuration, an intermediate temperature wherein analyte-binding oligos or other binders are in a configuration which is dependent upon analyte binding, and a high temperature wherein analyte-binding oligos or other binders are in a ‘loose’ or open configuration independent of analyte presence. Temperatures range from, for example, a low of 35, 40, 45, 50, 55, 60, 65 C, to a high of 40, 45, 50, 55, 60, 65, 70, 75, or 80 C. Other temperatures within and extending beyond these listed ranges are also contemplated as consistent with the disclosure herein.

[0148] Methods herein may assay, for example, at least 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000 or more analytes or analyte variants. Assay binding may in some cases be analyte concentration sensitive, such that some probes bind an analyte at different concentrations from one another. Similarly, some analyte binding signals may be indicative of analyte concentration as well as analyte presence, independent molecules.

[0149] In some cases, analyte detection is followed by or preceded by an assay for analyte binder identity such as an assay for an oligo tag indicative of identity of an analyte binder identity at a position. Alternately, in some cases reporter signal may be correlated to a location on an array such as a surface onto which a known analyte binder has been deposited. In other embodiments the temperature at which analyte binding dissociation occurs is indicative of target analyte identity at a position.

Definitions

[0150] A "small molecule" is defined herein to have a molecular weight below about 1000 Daltons, and is generally an organic compound. In some embodiments, a small molecule is an active agent or a prodrug or metabolite thereof. A small molecule may be charged or neutral. [0151] The singular forms "a" "an" and "the" include plural referents unless the context clearly dictates otherwise. Approximating language, as used in the description and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related.

[0152] As used herein, the term "about" in reference to a number refers to a range spanning from 10% less than the number to 10% greater than the number. Similarly, in reference to a range, the term "about" refers to an expanded range having a lower bound which is 10% less than the lower bound listed and an upper bound which is 10% greater than the upper bound listed.

[0153] As used herein, "stabilize," and its grammatical variants mean to hold steady or limit fluctuations. "Stabilizing" a complex results in promoting or prolonging the existence of the complex or inhibiting disruption of the complex including when the environment is changed (e.g. perturbation of the persistence of the binding competent state). The term can be applied to any of a variety of complexes including, but not limited to a binary complex. For example, the complex that is stabilized can be a binary complex between an immobilized molecule and a binding partner. Generally, stabilization of the binary complex increases the persistence of the binding competent state of the immobilized molecule upon a change in the presented environment to said immobilized molecule.

[0154] As used herein, a "nucleotide" is a molecule that includes a nitrogenous base, a five- carbon sugar (e.g., ribose, sulfo-ribose or deoxyribose), and at least one phosphate group including a phosphate ester when the nucleotide is part of a polynucleic acid, or functional analogs of such a molecule. Nucleotide analogs may optionally be without the 3 ’-OH group, replaced with a different moiety or modified with a moiety. In some embodiments, the moiety is a 3’ hydrogen or fluorine. The base of a nucleotide may be any of adenine, cytosine, guanine, thymine, or uracil, or analogs thereof. Optionally, a nucleotide has an inosine, xanthine, hypoxanthine, isocytosine, isoguanine, nitropyrrole (including 3 -nitropyrrole) or nitroindole (including 5-nitroindole) base. Nucleotides can include or exclude ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dUTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. In some embodiments, an aptamer can comprise a nucleotide which is intrinsically a fluorophore. In some embodiments, the nucleotides include SELEX-compatible nucleotides (or a variant thereof) or that can be introduced into a nucleic acid. In some embodiments, the SELEX-compatible nucleotides can include or exclude the following nucleotide modifications: substitution of 2'-OH by fluor (F), modification of 2'-OH by a methyl group (CH3), substitution of 2'-OH by an amino group (NH2), a Locked Nucleic Acid (LNA) with methylene bridge between 2'-0 and 4'-C, modification of C-5 by Bromine (Br), modification of C-5 by Iodine (I), and substitution of 4-0 by Sulfur (S). In some embodiments, the SELEX-compatible nucleotides can include or exclude: 2'-Fluoro-dUTP. 2'-Fluoro-dCTP, 2'-Fluoro-dATP, 2'-Fluoro-dGTP, 2'-Fluoro-dNTP, 2'0Me- UTP, 2'0Me-CTP, 2'0Me-ATP, 2'0Me-GTP, 2'NH2-dUTP, 2'NH2-dCTP, 2'NH2-dATP, 2'NH2-dGTP, LNA-ATP, LNA-GTP, LNA-CTP, LNA-UTP, 5-Bromo-dUTP, 5-Iodo-UTP, 4- Thio-UTP, s4UTP, and 4sUTP. In some embodiments, the SELEX-compatible nucleotides can be those described in Komarova et al., Molecules. 2019 Oct; 24(19): 3598, herein incorporated by reference. A SELEX-compatible nucleotide can be a nucleotide which can be included in a SELEX process without negatively impacting the ability of the SELEX process to arrive at an aptamer which can selectively bind to a target.

[0155] As used herein, "measuring" (or sometimes "detecting"), refers to a process of identifying the presence of a signal. For example, measuring may involve identifying fluorescence emitted from a fluorophore upon excitation with light. Measuring can be intermittent (e.g., periodic) or continuous (e.g., without interruption), and can involve acquisition of quantitative results. Measuring can be carried out by observing multiple signals over a period of time during the changing of the environment about the fluorophore (e.g., while the temperature of the system is increased or during the application of an electric field) or, alternatively, by observing signal(s) at a single time point during or after changing the environment around the fluorophore. In some embodiments, the measuring may occur before, during, or after the change in environment. In some embodiments, measuring can be continuously monitored over time as is typical of a time-based acquisition. It is also possible to acquire a series of time points in a periodic fashion to obtain a time-based acquisition. [0156] As used herein, "imaging" refers to a process for obtaining a representation of a sample or a portion thereof. The process may involve acquisition of optical data, such as the relative location of a feature undergoing analysis, and intensity of an optical signal produced at the position of the feature.

[0157] As used herein, "contacting," when used in reference to chemical reagents, refers to the mixing together of reagents (e.g., mixing an immobilized molecule and either a buffered solution that may include a binding partner) so that a physical binding reaction or a chemical reaction may take place.

[0158] As used herein, "biosensor" refers to a system comprising a "receptor" and an electronic sensor. The "receptor" binds a binding partner, which may include an analyte. In some embodiments, the "receptor" is an immobilized molecule as described herein.

[0159] As used herein, "electronic sensor" refers to an electronic transducer that converts photons to electrons. In particular, an electronic sensor converts the detection of a photon or photons into an electrical signal. An electronic sensor may be electrically connected to other computer circuit units which can include or exclude memory (transitory or permanent), central processing units, and graphic processing units. In some embodiments, an electronic sensor comprises a series of stacked layers which are in electronic communication with each other and wherein at least one of the stacked layers is photosensitive. While a "sensor" refers to a device, the terms "sensor molecule", and "biosensor molecule" refer to a modality.

[0160] In some embodiments, the immobilized molecule is a sensor molecule. Sensor molecules can be configured to be in a format where individual reactions (e.g., a binding reaction) can be isolated from another, and allows for the introduction of controlled perturbation. In some embodiments, the format can include or exclude flow cells, wells of a multiwell plate; microscope slides; tubes (e.g., capillary tubes), and beads in an emulsion. Features to be measured during changing the environment of an immobilized molecule can be contained within the isolated individual reactions. In some embodiments, the sensor molecule is connected to a solid support. In some embodiments, the sensor molecule is directly connected to a solid support by a covalent or non-covalent bond. In some embodiments, the sensor molecule is linked to the solid support by a linker. The linker can comprise a polymer. In some embodiments, the polymer is a hydrogel.

[0161] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in an aqueous liquid. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g., due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, beads, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefin copolymers, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, 2D materials (e.g., graphene), transparent conductive materials (e.g., Indium Tin Oxide, silver nanoparticle doped polymers), metals, inorganic glasses, mirrored surfaces, and polymers.

[0162] The solid support may take any of a variety of configurations ranging from simple to complex and can have any one of a number of shapes, including a strip, plate, disk, rod, particle, including bead, tube, well, and the like. The surface may be relatively planar (e.g., a slide), spherical (e.g., a bead), cylindrical (e.g., a rod), or grooved. Exemplary solid supports that may be used can include or exclude microtiter wells, microscope slides, membranes, paramagnetic beads, charged paper, Langmuir-Blodgett films, silicon wafer chips, flow through chips, and microbeads.

[0163] As used herein, "library" refers to a collection of species wherein not all of the species have the same identity.

[0164] As used herein, a "complex" refers to a molecular entity formed by covalent or non- covalent association involving two or more component molecular entities (e.g., an immobilized molecule and a binding partner). The complex is not necessarily transitory, in that the complex will remain as a bimolecular entity until subject to a change in environment. The complex forms because of biomolecular recognition between the immobilized molecule and the binding partner. [0165] As used herein, "equilibrium" generally refers to a state of balance due to the equal action of opposing forces (e.g., equal, opposite rates). For example, a complex formed between an immobilized molecule and a binding partner is in equilibrium with unbound immobilized molecule and binding partner when the rate of formation of the complex is balanced by the rate of its dissociation. Under this condition, the reversible binding reaction ceases to change its ratio of bound/unbound component. If the rate of a forward reaction (e.g., complex formation) is balanced by the rate of a reverse reaction (e.g., complex dissociation), then there is no net ratio change.

[0166] As used herein, "binding competent state" refers to the conformation or ensemble of conformations that an immobilized molecule adopts which can form a complex with a binding partner. In some embodiments, the conformation is a tertiary structure of the immobilized molecule ("binding competent conformation"). The binding competent state need not have a bound binding partner, but is a local energy minimum based on the structure of the immobilized molecule. In some embodiments, when the immobilized molecule is an aptamer, the binding competent state is the tertiary structure of the aptamer formed from the local energy minimum for folding. In some embodiments, aptamers in a binding competent state may comprise regions of double-strands, hairpin loops, and/or single strand sequences. In some embodiments, a cluster of immobilized molecules can comprise ensembles of binding competent states. For example, a first immobilized molecule in a cluster can comprise a first binding competent state, and a second immobilized molecule within the same cluster and having the same identity of the first immobilized molecule can comprise a second binding competent state. In another example, a cluster can be formed from two different aptamers that each recognize different parts of the same analyte, such that avidity favors the selective binding of the analyte. Complex formation can involve conformational changes as a prerequisite for binding (e.g., conformational selection) or concurrently with binding (e.g., induced-fit). Regardless, the stability/persistence of the binding competent ensemble of states will change upon association with a binding partner. [0167] The term "sample" as used herein refers to an aliquot of material, frequently an aqueous solution or an aqueous suspension derived from biological material. In some embodiments, the sample can be a biological sample. The biological sample can be from a living subject. For example, in some embodiments, the sample may be any sample containing cells. In some embodiments, the sample may be from, for example, whole blood, bone marrow, serum, plasma, cerebrospinal fluid, sputum, bronchial washings, bronchial aspirates, urine, lymph fluids and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, sweat, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants, tissue specimens which may or may not be fixed, and cell specimens which may or may not be fixed, or a fine needle aspirate. Samples to be assayed for the presence of an analyte by the methods of the present invention include, for example, cells, tissues, homogenates, lysates, extracts, purified or partially purified proteins and other biological molecules and mixtures thereof. In some embodiments, the biological sample may be processed. The processing can be, for example, removal of selected species in the sample.

[0168] As used herein, "subject" refers to any source of biological or nonbiological sample for which an analyte is to be assayed. In some cases the subject is a mammal that can include or exclude humans, domestic and farm animals, and zoo, or pet animals, such as dogs, horses, cats, mouse, rat, rabbits, monkeys, llama, sheep, pigs, cows, etc., or exotic or 'wild' animals such as bats, wild animals caught for sale, or wild animals (for example, one suspected of harboring a pathogen capable of impacting humans). The preferred mammal herein is a human, including adults, children, and the elderly. In some embodiments, the subject is an aquatic park animal, such as a dolphin, whale, seal or walrus. A subject can also include any organism used in clinical or preclinical trials. Alternately, a subject may comprise an environmental sample, such as an environmental sample suspected of harboring an organism or analyte of interest.

[0169] A sample drawn from a subject may be assayed subsequent to processing, such as buffering, stabilization, or purification of the sample by, for example, purification or enrichment of proteins, small molecules such as hormones or starches, nucleic acids such as DNA or RNA, or lipids, cells, viruses, or other molecule or molecule classes in the sample, removal of proteins, small molecules such as hormones or starches, nucleic acids such as DNA or RNA, or lipids, cells, viruses, or other molecule or molecule classes from the sample. Alternately, a sample may be assayed raw, without subjecting the sample to selective enrichment, buffering or stabilization. Furthermore, subsequent to contacting a sample to a binding agent, the sample and binding agent, surface, beads or other binding vicinity may be subjected to a wash step. The wash step may serve to remove constituents of the sample that may interfere with the assay, by for example blocking binding or leading to background fluorescence that may impact signal detection, or may remove nonspecifically bound sample constituents. Other wash functions are consistent with the disclosure herein.

[0170] As used herein, the term "binding partner" refers to any known or unknown substance that can be recognized by the immobilized molecule. The term "binding partner" may include, for example, ions, small molecules (e.g., having a molecular weight of less than 1000 Da), proteins, peptides, glycoproteins, cells, cell-surface molecules or proteins or glycoproteins, viruses, organelles, synthetic polymers, carbohydrates, hormones, cytokines, growth factors, toxins, cell surface receptors, bacterial or parasitic cell components, or viral antigens, or a component thereof In some embodiments, a binding partner may be obtained from a sample comprising complex mixture of ions, small molecules (e.g., having a molecular weight of less than 1000 Da), proteins, peptides, glycoproteins, a cell, a cell-surface molecule or protein or glycoprotein, a virus, an organelle, a synthetic polymer, a carbohydrate, or a component thereof. In some embodiments, when the binding partner is a cell, the cells may be a transformed cell which can be transfected with an oncogene which is integrated into the cell. In some embodiments, the transformed cells may include or exclude, for example, mammalian cells, immunomodulatory cells, leukocytes, tumor cells, yeast cells, bacterial cell, infectious agents, parasites, plant cells, transfected cells such as NSO, CHO, COS, 293 cells. Transformation of cells such as NSO, CHO, COS and 293 cells can be achieved by a method which can include or exclude electroporation and nucleofection. In some embodiments, the binding partner can be present on the cell surface, within the cell, or both on the surface and within the cell. In some embodiments the binding partner may be present in or on one or more cellular features, for example, the cytosol, the nucleus, the nuclear membrane, nucleoli, the endoplasmic reticulum, Golgi apparatus or mitochondria.

[0171] In some embodiments, both the binding partner and the immobilized molecule are not nucleic acids (e.g., DNA microarray). When the binding partner is a nucleic acid, the immobilized molecule is often not a nucleic acid. When the immobilized molecule is a nucleic acid, the binding partner is often not a nucleic acid. The terms "specifically binding" and "specific binding" as used herein mean that a protein (e.g., antibody or lectin), aptamer, or other immobilized molecule of interest, binds to a target such as an antigen, ligand or other analyte, with a different affinity than it binds to other molecules under the specified conditions of the present invention, such that under certain conditions the molecule of interest can be said to be specifically bound while other molecules are not bound by the binding moiety.

[0172] As used herein, "environment" refers to the surroundings or conditions to which the immobilized molecule is exposed. An environment may include or exclude contributions from an electric field, a magnetic field, a thermal energy, a gravitational field, flow rate, shear rate, light intensity or wavelength or polarization, ionic strength, or chaotropic agent concentration. In some embodiments, one element of an environment may be present while the others are absent. For example, the ionic strength may be a selected buffer concentration but the chaotropic agent concentration is zero. An environment may be changed by the application of an external force. The applied external force can include or exclude: electric field intensity, electric field direction, magnetic field, gravitational field, shear force, increased or decreased temperature, light polarization change, light intensity change, light wavelength change, increase or decrease in an ion or chaotropic agent concentration. In some embodiments, an environment change may result in the denaturation of the immobilized molecule. In some embodiments, an environmental change may result in the folding or unfolding of an immobilized molecule (e.g., protein or nucleic acid). In some embodiments, the environmental change may result in a conformational change of the immobilized molecule. Exemplary environmental changes include temperature, ion concentration, voltage, current or electrical charge, or any other permutation that may impact analyte binding or analyte binding oligo or other analyte binding moiety configuration.

[0173] As used herein, the term "chaotropic agent" includes its commonly understood meaning in the field and refers to agents such as guanidinium hydrochloride or urea, which disrupt hydrogen bonds to potentially destabilize the binding competent states of biomolecules.

[0174] As used herein, the term "electric field" refers to a field generated by the presence of a voltage gradient which exerts a force on a point charge or a force on a multipole. The intensity of an electric field can be modulated resulting in a corresponding change in the applied force upon a charged particle or multipole subject to the electric field. An electric field is a vector, in that there is also a directional element. In some embodiments, the magnitude and/or direction of an electric field may be changed to perturb the environment upon which an immobilized molecule is subject to.

[0175] As used herein, a "kit" is a packaged unit containing one or more components that can be used for performing detection of analytes. Typical kits may include packaged combinations, in one or more containers or vials of reagents, a consumable cartridge, configured to be used in the methods described herein.

[0176] As used herein, the term "antibody" refers to an immunoglobulin or fragment thereof that can specifically bind to an antigen (binding partner). In some embodiments, an antibody can include or exclude any recombinant or naturally occurring immunoglobulin molecule such as a member of the IgG class, (e.g., IgGl), antibody fragment, ScFv (single-chain variable fragment), a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins (optionally wherein the fusion VH and VL chains are connected with a short linker peptide of ten to about 25 amino acids), or single-domain antibody (nanobody), and any derivatives thereof. In some embodiments, the antibody can be a monoclonal or polyclonal antibody.

[0177] The term "antibody fragments" as used herein, refers to a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In some embodiments, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind an antigen. An antibody fragment can include or exclude Fv, Fab and F(ab’)2 fragments.

[0178] "Polyclonal Antibodies" or "PAbs," are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as rabbits, mice and goats, may be immunized by injection with an antigen or antigen-conjugate, optionally supplemented with adjuvants. Polyclonal antibodies may be unpurified, purified or partially purified from other species in an antiserum. The techniques for the preparation and purification of polyclonal antibodies are described in various general and more specific references, including but not limited to Kabat & Mayer, Experimental Immunochemistry, 2d ed., (Thomas, Springfield, Ill. (1961)); Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)); and Weir, Handbook of Experimental Immunology, 5th ed. (Blackwell Science, Cambridge, Mass. (1996)).

[0179] "Monoclonal antibodies," or "MAbs," are homogeneous populations of antibodies to a particular antigen and may be obtained by any technique that provides for the production of antibody molecules, such as by continuous culture of cell lines. These techniques include, but are not limited to the hybridoma technique of Kohler and Milstein, Nature, 256:495-7 (1975); and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor, et al., Immunology Today, 4:72 (1983); Cote, et al., Proc. Natl. Acad. Sci. USA, 80:2026-30 (1983)), and the EBV-hybridoma technique (Cole, et al., in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96 (1985)). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the MAb of this invention may be cultivated in vitro or in vivo. Production of high titers of MAbs in vivo makes this a presently preferred method of production.

[0180] Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-26 (1988); Huston, et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988); and Ward, et al., Nature, 334:544-46 (1989)) can be adapted to produce single chain antibodies suitable for use in the present invention. Single chain antibodies are typically formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

[0181] Antibody fragments include but are not limited to: the F(ab’)2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab’)2 fragments. Alternatively, Fab expression libraries may be constructed (Huse, et al., Science, 246: 1275-81 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[0182] The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Techniques developed for the production of "chimeric antibodies" (Morrison, et al., Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Takeda, et al., Nature, 314:452-54 (1985)) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody can be a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine MAb and a human immunoglobulin constant region. [0183] The terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may comprise deoxyribonucleotides, ribonucleotides and/or their analogs. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown. A nucleic acid molecule may also comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5 -fluorouracil, 5 -bromouracil, 5- carboxymethylaminomethyl-2 -thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6- isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.

[0184] Sugar modifications (e.g., 2-O-methyl, 2-fluor and the like) and phosphate backbone modifications (e.g., morpholino, locked nucleic acid (LNA), unlocked nucleic acid, peptide nucleic acid (PNA), thioates, dithioates, phosphorothiolates, phosphorothioates, methyl phosphonates, and the like) can be incorporated singly, or in combination, into the nucleic acid molecules of the present invention. In some embodiments, for example, a nucleic acid of the invention may comprise a modified sugar and a modified phosphate backbone. In another embodiment, a nucleic acid of the invention may comprise modifications to sugars, bases, and/or phosphate backbone.

[0185] The nucleotide sequence of the aptamer nucleic acids of the present invention is of less importance than the functional roles they are required to perform. Accordingly, the sequence of the aptamer nucleic acids, and the length of the aptamer nucleic acid, may vary considerably, provided the aptamer nucleic acid can still perform the functional roles they are required to perform. Importantly, the sequence and length of the aptamer nucleic acids are not limited to those exact sequences and lengths of the exemplary binding pairs disclosed herein. The aptamer nucleic acids thus can be of different lengths and or sequence, and vary in identity and/or length to the disclosed aptamer nucleic acids. In some embodiments, the aptamer nucleic acids can have 80, 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to those aptamer sequences disclosed herein. An important function of the aptamer nucleic acid of the present invention is to provide a binding competent state to the binding partner to form a complex.

[0186] As used herein, "aptamer" refers to a nucleic acid that binds to a target (a binding partner). In some embodiments, the binding is specific binding for a target molecule, such target molecule having a three-dimensional chemical structure, other than a polynucleotide, that binds to the aptamer through a mechanism which is predominantly independent of Watson/Crick base pairing or triple helix binding. In some embodiments, the aptamer can be one such as those described in S. Lapa, et al., Molecular Biotechnology volume 58, p. 79-92 (2016); or S. Gao, et al., Analytical and Bioanalytical Chemistry volume 408, p. 4567-4573 (2016)), herein incorporated by reference. Aptamers include nucleic acids that are identified from a candidate mixture of nucleic acids by the process referred to as SELEX and variations thereof. In some embodiments, the aptamer is developed by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture may be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield a ligand- enriched mixture of nucleic acids, whereby aptamers to the target molecule are identified. Affinity interactions may vary in degree; however, in this context, the "specific binding affinity" of an aptamer for its target means that the aptamer binds to its target generally with a higher degree of affinity than it may binds to other, non-target, components in a mixture or sample. A "cluster of aptamers" is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence and are grouped together in a defined location on a surface. An aptamer can include any suitable number of nucleotides. Different aptamers may have either the same number or a different number of nucleotides. Aptamers may comprise or be DNA or RNA or variants thereof, and may comprise single stranded, double stranded, and/or hairpin regions. [0187] In some embodiments, the nucleic acid composition of an aptamer can be varied to produce an aptamer with a selected persistence upon self-folding into a binding competent state or to optimize its affinity for its target. In some embodiments, the modified nucleic acid in the aptamer can include or exclude: peptide-nucleic acids (PNA), locked nucleic acids (LNA), or normal deoxyribonucleic acid (DNA). PNAs have a peptide-backbone rather than a ribosephosphate backbone of normal DNA. The PNA backbone is composed of repeating N-(2- aminoethyl)-glycine units linked by peptide bonds. The purine and pyrimidine bases are linked to the PNA backbone by a methylene bridge (-CH2-) and a carbonyl group (-(C=O)-), The PNA backbone thus lacks charged phosphate groups. PNAs are not easily recognized by either native nucleases or proteases, imbuing them resistance to enzymatic degradation and pH stability. The LNA backbone comprises a ribose moiety which is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon locking the ribose in the 3-endo (North) conformation. The locked ribose conformation enhances base stacking and backbone pre-organization and significantly increases the duplex stability of LNA/DNA duplexes. Methyl phosphonate backbones replace the charged anionic phosphate with a neutral methyl phosphonate ester. Thiophosphonate backbones comprise non-bridging oxygen on the phosphate backbone to form a phosphorothioate (PS) linkage. Thiophosphonate backbones exhibit nuclease resistance should a nuclease be present in the sample. Not all of these constituent nucleotides can be included in SELEX, but can be used to modify a nucleic acid molecule.

[0188] In some embodiments, the aptamer can further comprise one or a plurality of non-natural nucleotides. In some embodiments, the non-natural nucleotide can be iso-G or iso-C (or derivatives thereof), as described in Richert, C., et al. J. Am. Chem. Soc. 118, 4518-4531 (1996), herein incorporated by reference. In some embodiments, the non-natural nucleotide can be diflurotoluene (or derivatives thereof), as described in Schweitzer, B. A., et al., J. Am. Chem. Soc. 117, 1863-1872 (1995), herein incorporated by reference. In some embodiments, the non- natural nucleotide can be MM02 or SICS (or derivatives thereof), as described in Leconte, A. M. et al. J. Am. Chem. Soc. 130, 2336-2343 (2008), herein incorporated by reference. In some embodiments, the non-natural base can be Ds or Diol-Px (or derivatives thereof), as described in Yamashige, R. et al. Nucl. Acids Res. 40, 2793-2806 (2012), herein incorporated by reference. In some embodiments, the non-natural nucleotide can be P or Z (or derivatives thereof), as described in Yang; Z., et al., J. Am. Chem. Soc. 133, 15105-15112(2011), herein incorporated by reference. In some embodiments, the non-natural nucleotide can be NaM or 5 SICS (or derivatives thereof), as described in Malyshev, D. A. et al. Proc. Natl Acad. Sci. USA 109, 12005-12010 (2012), herein incorporated by reference. In some embodiments, the non-natural nucleotide can be of an expanded genetic code as described in Kimoto et al., Chem. Soc. Rev., 2020,49, 7602-7626, herein incorporated by reference. In some embodiments, the aptamers comprising one or a plurality of non-natural nucleotides can be modified before, or after SELEX identification so as to include a non-natural nucleotide.

[0189] As used herein, "immobilized molecule" refers to a molecule such as an analyte binding probe or other analyte binder which is located at a particular region such that it may be located subsequent to detection. In some embodiments, a molecule is immobilized as a result of it being connected to a solid support such that the immobilized molecule may not translocate on or off the solid support. In some embodiments, the immobilized molecule is a synthetic polymer or a biomolecule ("immobilized biomolecule"). Examples of the immobilized biomolecule can include or exclude an ion, a small molecule, antibody, aptamer, protein, lectin, aptamer, carbohydrate (e.g., sugar or oligosaccharide), or peptide. In some embodiments, the immobilized biomolecule includes an immobilized biosensor biomolecule (immobilized sensor biomolecule). Methods or chemistries suitable for immobilization are known in the art.

[0190] Alternately, in some cases a molecule is immobilized by being localized to a confined volume, such as a well or an emulsion droplet. In these cases, the molecule is not tethered but is located at a particular region such that it may be located subsequent to detection. [0191] As used herein, "energy transfer relationship" refers to a relationship between two dyes (e.g., a "donor" and an "acceptor") held sufficiently close that energy emitted by one dye can be received or absorbed by the other dye. The "donor" is the moiety that initially absorbs the energy, and the "acceptor" is the moiety to which the energy is subsequently transferred.

[0192] As used herein, "FRET" (i.e., Forster resonance energy transfer) refers to the distancedependent transmission of energy from the site of absorption to the site of its utilization (e.g., fluorescence) in a molecule or system of molecules by resonance interaction between chromophores.

[0193] Turning to the Figures, one sees the following.

[0194] At Figure 1, one sees a probe complexed to an analyte detection oligo. The probe, at left, comprises BHQ1 covalently attached to the 5’ end of a first segment, FAM which is covalently attached to the 3 ’ end of a second segment, and a linker connecting the first and second segments. BHQ1 and FAM together comprise the two-part reporter. Their activity is mediated by their being in physical proximity.

[0195] The analyte-binding oligo is at right. It comprises both a central target specific protein binding site as well as 5’ and 3’ end configuration-reporting moi eties that are common to some or all of the analyte binders in an assay.

[0196] The 5’ and 3’ end configuration-reporting moi eties are bound to the first segment and the second segment of the probe. The target specific protein binding site is bound to its target analyte, which stabilizes the analyte-binding oligo in a ‘tight’ configuration such that the 5’ and 3’ end configuration-reporting moi eties are held in a configuration which reports binding to the analyte.

[0197] Although not indicated directly in the figure, implicit in the depiction is that the complex is at a temperature at which analyte binding is able to stabilize the analyte-binding oligo in the tight configuration. At temperatures above a threshold analyte binding is abolished and the analyte binding oligo assumes a ‘loose’ or ‘open’ configuration such that the 5’ and 3’ end configuration-reporting moi eties are not held in a configuration that allows a bound probe BHQ1 quencher to repress signaling by a FAM fluorophore in a split reporter moiety system.

[0198] Also not shown is an optional tether of the analyte-binding oligo to a surface. Such binding may be effected at the analyte-binding oligo 5’ end, 3’ end or at an internal position. [0199] In some cases, the 5’ and 3’ end configuration-reporting moi eties correspond to primers or primer binding sites used in a SELEX process from which the target binding region was developed.

[0200] At Fig. 2, one sees an alternative probe complexed to an analyte detection oligo. The probe, at left, comprises FAM covalently attached to the 3’ end of a first segment and DBCO which is covalently attached to the 3’ end of the first segment. BHQ1 and FAM together comprise the two-part reporter. Their activity is mediated by their being in physical proximity. [0201] The analyte-binding oligo is at right. It comprises both a central target specific protein binding site as well as a 3’ end configuration-reporting moiety that works in combination with a 5’ covalently bound BHQ1 to report binding status or configuration status.

[0202] The 5’ BHQ1 quencher is unbound, while the 3’ end configuration-reporting moiety of the analyte-binding oligo is bound to the first segment of the probe. The target specific protein binding site is bound to its target analyte, which stabilizes the analyte-binding oligo in a ‘tight’ configuration such that the 5’ BHQ1 and 3’ end configuration-reporting moiety are held in a configuration which brings the FAM of the bound probe in proximity so as to report binding to the analyte.

[0203] Although not indicated directly in the figure, implicit in the depiction is that the complex is at a temperature at which analyte binding is able to stabilize the analyte-binding oligo in the tight configuration. At temperatures above a threshold analyte binding is abolished and the analyte binding oligo assumes a ‘loose’ or ‘open’ configuration such that the 5’ and 3’ ends are not held in a configuration that allows a bound probe BHQ1 quencher to repress signaling by a FAM fluorophore in a split reporter moiety system.

[0204] The DBCO acts as a tether holding the probe to a surface. DBCO may be added to the probe or to the analyte binding oligo or other analyte binder.

[0205] In some cases, the analyte binding oligo 3’ end .and the probe 5’ end comprise unique or specific reverse complementary sequence such that binding between the probe and the analytebinding oligo is specific. In these cases, the expected position of a particular analyte-binding oligo or other moiety is specified by the location of the complementary probe on a surface.

[0206] Alternately, when the probe is surface-tethered or tethered to a bead, decoding approaches may be used to identify the bound analyteObinding moiety, such as those in Gunderson 2004 or elsewhere disclosed or referred to herein or otherwise known in the art.

[0207] At Fig. 3, one sees results of a control assay using a fluorophore-quencher labeled analyte binding oligo detection system. Unlike the technology disclosed herein, the fluorophore is attached to the analyte biding oligo in this assay. At left, fluorescence is given for each of 8 concentrations of the target analyte, 0, 3, 6, 15, 25, 50, 100 and 200 nM, for a temperature gradient ranging from 30 to 90 C in 10 C intervals across the x axis. One sees that Fluorescence is delayed until after 60 C upon addition of 200 nM target, indicating that the target stabilized the quencher near the fluorophore at this analyte concentration, consistent with analyte binding. 100 nm analyte showed intermediate results. At right one sees the negative derivative of the curves shown at left. One sees again that the change in slope for the 200 nM analyte assay occurs above 60 C, in contrast to concentrations of 50 nM and below, where the change in slope occurs at about 50 C. 100 nM concentration shows intermediate behavior. These results indicate that the analyte stabilizes the fluorophore near the quencher starting at concentrations of 100 nM, resulting in quenching up to above 60C.

[0208] At Fig. 4, one sees results of a reaction using a fluorophore-quencher labeled probe to assay an unlabeled analyte binding oligo detection system. The fluorophore is attached to the probe in this assay and the analyte binding oligo is unlabeled. At left, fluorescence is given for each of 8 concentrations of the target analyte, 0, 3, 6, 15, 25, 50, 100 and 200 n<, for a temperature gradient ranging from 30 to 90 C in 10 C intervals across the x axis. One sees that Fluorescence is delayed until after 60 C upon addition of 200 nM target, indicating that the target stabilized the quencher near the fluorophore at this analyte concentration, consistent with analyte binding. 100 nm analyte showed intermediate results. At right one sees the negative derivative of the curves shown at left. One sees again that the change in slope for the 200 nM analyte assay occurs above 60 C, in contrast to concentrations of 50 nm and below, where the change in slope occurs at about 50 C. 100 nM concentration shows intermediate behavior. These results indicate that the analyte stabilizes the fluorophore near the quencher starting at concentrations of 100 nM, resulting in quenching up to above 60C.

[0209] At Fig. 5, one sees distance calculations associated with the measurements in Fig. 3 (control) and Fig. 4 (eGlint arm). The Y axis represents distance from 0 to 100 in intervals of 20. The X axis represents the target analyte, Platelet Derived Growth Factor, on a logarithmic scale ranging from lOeO to 10e2. One sees that the control and the probe system disclosed herein exhibit similar dose-dependent response as evidenced by comparable distance calculations across the range of concentrations tested.

EXAMPLES

Example 1. Analyte detection using a probe-labeled reporter system.

[0210] Unlabeled analyte-binding oligos assayed using a fluorophore-quencher labeled visualization dye were compared in their performance to a fluorophore-quencher labeled analyte binding probe in the detection of platelet derived growth factor. Analytes were provided at the concentrations indicated and the compositions were subjected to a continuous temperature gradient from below 30 °C to over 90 °C, during which fluorescence is measured and the change in fluorescence over time is calculated. Generally, a sharp increase in fluorescence indicates a loss of analyte binding complex structure, resulting in the fluorophore and quencher no longer being held in proximity. Shift to a higher temperature at which this loss of structure occurs is indicative of stabilization of the structure through analyte binding. [0211] The results are presented in Fig. 3 - Fig. 4. The results indicate that moving the fluorophore-quencher system from the analyte binding oligo to the probe does not disrupt analyte detection capacity, and that unlabeled analyte binding oligos assayed using fluorophore- quencher labeled probes effectively detect analytes. An analyte binding oligo the systems herein shows effective, repeatable detection of an analyte.

[0212] Distance values were calculated for the experimental probes and for a control. One sees that experimental arms and controls exhibited comparable distances across a broad range of analyte concentrations. The results are depicted in Fig. 5.

Claims

CLAIMS What is claimed is as follows:

1. A system for detecting an analyte, comprising a first oligonucleotide comprising an analyte specific binding region bounded by a first universal region and a second universal region; a second oligonucleotide segment comprising a first universal region reverse complement region and a paired fluorophore; and a third oligonucleotide segment comprising a second universal region reverse complement region and a paired fluorophore complement.

2. The system of claim 1, wherein the analyte specific binding region comprises an analyte binding site.

3. The system of claim 1, wherein the analyte specific binding region assumes a compact configuration when bound to the analyte.

4. The system of claim 3, wherein the compact configuration brings the first universal region and the second universal region in closer proximity upon analyte binding relative to the first universal region and the second universal region proximity in the absence of analyte binding.

5. The system of claim 1, wherein the first oligonucleotide analyte specific binding region binds the analyte such that the first universal region and the second universal region are brought in closer proximity upon analyte binding relative to the first universal region and the second universal region proximity in the absence of analyte binding.

6. The system of claim 1, wherein the first oligonucleotide does not comprise a fluorophore.

7. The system of claim 1, wherein the analyte specific binding region is tethered to the first universal region by a flexible linker.

8. The system of claim 7, wherein the flexible linker comprises at least on thymidine (T) base.

9. The system of claim 1, wherein the analyte specific binding region is tethered to the second universal region by a flexible linker.

10. The system of claim 9, wherein the flexible linker comprises at least on thymidine (T) base.

11. The system of claim 1, wherein the first oligonucleotide is tethered to a solid surface.

12. The system of claim 11, wherein the tether is at the first oligonucleotide 5’ end.

13. The system of claim 11, wherein the tether is at the first oligonucleotide 3’ end.

14. The system of claim 11, wherein the tether is at an internal position on the first oligonucleotide.

15. The system of claim 11, wherein the solid surface is a locally planar surface.

16. The system of claim 11, wherein the solid surface is a bead.

17. The system of claim 1, wherein the first universal region and the second universal region arise from polymerase chain reaction of the first oligonucleotide analyte specific binding region.

18. The system of claim 1, wherein the first oligonucleotide analyte specific binding region is generated through SELEX selection.

19. The system of claim 1, wherein the second oligonucleotide segment and the third oligonucleotide segment share a common phosphodiester backbone.

20. The system of claim 1, wherein the second oligonucleotide segment and the third oligonucleotide segment do not share a common phosphodiester backbone.

21. The system of claim 1, wherein the second oligonucleotide segment is not tethered to a solid surface.

22. The system of claim 1, wherein the paired fluorophore comprises BHQ1.

23. The system of claim 1, wherein the paired fluorophore complement comprises FAM.

24. The system of claim 1, wherein the paired fluorophore and the paired fluorophore complement comprise a FRET pair.

25. The system of claim 1, wherein the analyte does not comprise a nucleic acid.

26. The system of claim 1, wherein the analyte comprises a protein.

27. The system of claim 26, wherein the protein is a cell surface protein.

28. The system of claim 1, wherein the analyte comprises a viral particle.

29. The system of claim 1, wherein the analyte comprises an epitope.

30. A method of high throughput sample analysis, comprising contacting a sample to a plurality of analyte-specific oligos that share common bipartite universal detection oligo binding sites, and assaying for binding of at least one of the plurality of analyte-specific oligos to its analyte by contacting a plurality of analyte-specific oligos to a universal detection oligo population.

31. The method of claim 30, wherein assaying for binding comprises assaying for binding- activated fluorescence of at least one oligo of the universal detection oligo population.

32. The method of claim 30, wherein contacting an analyte of the sample to an analytespecific oligo of the plurality of analyte-specific oligos that binds the analyte brings the common bipartite universal detection oligo binding sites of the analyte-specific oligo into a binding configuration such that they are in closer proximity to one another.

33. The method of claim 32, wherein fluorescence of the oligos of the universal detection oligo population is differentially activated by binding to universal detection oligo binding sites of the analyte-specific oligos in the binding configuration.

34. The method of claim 30, wherein the plurality of analyte-specific oligos are tagged using distinguishing tags such that a signal from an analyte specific oligo bound to its analyte locations.

35. The method of claim 34, wherein the distinguishing tags are barcodes.

36. The method of claim 34, wherein the distinguishing tags are sequence templates.

37. The method of claim 34, wherein the distinguishing tags are identifier probe binding sites.

38. The method of claim 30, wherein the plurality of analyte-specific oligos are tethered to a surface.

39. The method of claim 38, wherein the plurality of analyte-specific oligos are tethered in array such that detecting a signal from a position on the array may be correlated to a known oligo of the plurality of analyte-specific oligos.

40. The method of claim 38, wherein the plurality of analyte-specific oligos are tethered in array such that detecting a signal from a position on the array may be correlated to binding of a known analyte to a known oligo of the plurality of analyte-specific oligos.

41. The method of claim 38, wherein the plurality of analyte-specific oligos are tethered in array such that detecting a signal from a position on the array may be correlated to presence of a known analyte.

42. The method of claim 30, wherein the sample is an unprocessed sample.

43. The method of claim 30, wherein the sample is a processed sample.

44. The method of claim 30, wherein the sample comprises a protein analyte.

45. The method of claim 30, wherein the sample comprises at least 10 analytes.

46. The method of claim 30, wherein the sample comprises at least 100 analytes.

47. The method of claim 30, wherein the sample comprises at least 1000 analytes.

48. The method of claim 30, wherein the plurality of analyte-specific oligos comprise oligos having distinct analyte specific binding domains and share common universal detection oligo binding sites.

49. The method of claim 48, wherein at least some of the distinct analyte specific binding domains bind to distinct analytes at a common temperature.

50. The method of claim 48, wherein at least some of the distinct analyte specific binding domains bind to distinct analytes at distinct temperatures.

51. The method of claim 48, wherein the plurality of analyte-specific oligos comprise at least 50 types of oligos having distinct analyte specific binding domains from one another.

52. The method of claim 48, wherein the plurality of analyte-specific oligos comprise at least 100 types of oligos having distinct analyte specific binding domains from one another.

53. The method of claim 48, wherein the plurality of analyte-specific oligos comprise at least 200 types of oligos having distinct analyte specific binding domains from one another.

54. The method of claim 48, wherein the plurality of analyte-specific oligos comprise at least 500 types of oligos having distinct analyte specific binding domains from one another.

55. The method of claim 48, wherein the plurality of analyte-specific oligos comprise at least 1000 types of oligos having distinct analyte specific binding domains from one another.

56. The method of claim 30, wherein the plurality of analyte-specific oligos are not covalently bound to one or more fluorophores.

57. The method of claim 48, wherein the common universal detection oligo binding sites do not differ among the plurality of analyte-specific oligos.

58. The method of claim 48, wherein the common universal detection oligo binding sites do not specifically bind to an analyte of the sample.

59. The method of claim 48, wherein the common universal detection oligo binding sites correspond to polymerase amplification primer sites.

60. The method of claim 30, wherein the universal detection oligo population comprises a uniform oligo population.

61. The method of claim 60, wherein oligos of the universal detection oligo population bind to the common universal detection oligo binding sites of the plurality of analyte-specific oligos.

62. The method of claim 30, wherein oligos of the universal detection oligo population are labeled using a bipartite proximity fluorophore system.

63. The method of claim 62, wherein the bipartite proximity fluorophore system comprises BHQ1 and FAM.

64. The method of claim 62, wherein the bipartite proximity fluorophore system comprises a FRET fluorophore pair.

65. The method of claim 62, wherein the bipartite proximity fluorophore system comprises a spit fluorescent protein pair.

66. A surface comprising: a first oligo comprising a first analyte binding region and a first adapter and a second adapter, wherein the first oligo is tethered to the surface and bound to a first analyte such that the first adapter and the second adapter are in local proximity, and wherein the first oligo is bound to a first adapter binding region and a second adapter biding region, such that the first adapter binding region and the second adapter binding region are held in proximity.

67. The surface of claim 66 comprising a second oligo comprising a second analyte binding region and a first adapter and a second adapter, wherein the second oligo is tethered to the surface and not bound to an analyte such that the first adapter and the second adapter are not in local proximity, and wherein the first oligo is bound to a first adapter binding region and a second adapter biding region, such that the first adapter biding region and the second adapter binding region are not held in proximity.

68. The surface of claim 66 or claim 67, wherein the first adapter region and the second adapter region share a common phosphodiester backbone.

69. The surface of claim 66 or claim 67, wherein the first adapter region and the second adapter region do not share a common phosphodiester backbone.

70. The surface of any one of claims 66 to 69, wherein the first adapter region comprises a split fluorophore first part and wherein the second adapter region comprises a split fluorophore second part, such that that the split fluorophore may be active when first adapter biding region and the second adapter binding region are held in proximity.

71. The surface of any one of claims 66 to 70, wherein the first oligo is tethered at a first position such that fluorescence emitted from the first position can be correlated to the first oligo.

72. The surface of cany one of claims 67 - 71, wherein the second oligo is tethered at a second position such that fluorescence emitted from the second position can be correlated to the second oligo.

73. The surface of any one of claims 66 to 70, wherein the first oligo is tethered at a first position such that fluorescence emitted from the first position can be correlated to detection of the first analyte.

74. The surface of cany one of claims 67 - 71, wherein the second oligo is tethered at a second position such that absence of fluorescence emitted from the second position can be correlated to absence of the second analyte.

75. A surface comprising unlabeled specific analyte probes, and capable of being bound at the unlabeled specific analyte probes to labeled nonspecific adapter probes, wherein binding of at least one of the unspecific analyte probes causes reconfiguration of at least one of the labeled nonspecific adapter probes such that a label of the at least one of the labeled nonspecific adapter probes may be detected.

76. The surface of claim 75, wherein the unlabeled specific analyte probes are bound to the surface in an array such that fluorescence emitted from a first position on the surface can be correlated to binding of a first unlabeled specific analyte probe to a first analyte.

77. The surface of claim 76, wherein absence of fluorescence from a second position on the surface can be correlated to absence of binding of a second unlabeled specific analyte probe to a second analyte.

78. A method of detecting a plurality of analytes without requiring covalently labeled analyte probes, comprising contacting a sample to a plurality of analyte probes, wherein the analyte probes undergo a conformational change to an analyte binding configuration in response to binding to their respective analytes in the sample, and binding the plurality of analyte probes to a population of universal labeled probes, wherein the universal labeled probes emit a signal only when the analyte probes are in an analyte binding configuration.

79. The method of claim 78, wherein the plurality of analyte probes are tethered to a surface in an array such that a signal from a position on the surface can be correlated to an analyte probe directed to a known analyte at the position in the array.

80. The method of claim 78, wherein the universal labeled probes do not specifically bind an analyte.

81. The method of claim 78, wherein the universal labeled probes do not specifically bind a probe of the analyte probes.

82. The method of claim 78, wherein the universal labeled probes do not specifically bind analyte probes in an analyte binding configuration relative to analyte probes not in an analyte biding configuration.

83. A system for detecting an analyte, comprising a first oligonucleotide comprising an analyte specific binding region bounded by a first universal region and reporter system first component; a second oligonucleotide segment comprising a first universal region reverse complement region and a reporter system second component.

84. The system of claim 83, wherein the analyte specific binding region comprises an analyte binding site.

85. The system of claim 83, wherein the analyte specific binding region assumes a compact configuration when bound to the analyte.

86. The system of claim 85, wherein the compact configuration brings the first universal region and reporter system first component in closer proximity upon analyte binding relative to the first universal region and reporter system first component proximity in the absence of analyte binding.

87. The system of claim 83, wherein the first oligonucleotide analyte specific binding region binds the analyte such that the first universal region and reporter system first component are brought in closer proximity upon analyte binding relative to the first universal region and reporter system first component proximity in the absence of analyte binding.

88. The system of claim 83, wherein the first oligonucleotide does not comprise a fluorophore.

89. The system of claim 83, wherein the first oligonucleotide does not comprise a quencher.

90. The system of claim 83, wherein the first oligonucleotide is tethered to a solid surface.

91. The system of claim 83, wherein the second oligonucleotide is tethered to a solid surface.

92. The system of claim 90 or claim 91, wherein the tether is at the first oligonucleotide 5’ end.

93. The system of claim 90 or claim 91, wherein the tether is at the first oligonucleotide 3’ end.

94. The system of claim 90 or claim 91, wherein the tether is at an internal position on the first oligonucleotide.

95. The system of claim 90 or claim 91, wherein the solid surface is a locally planar surface.

96. The system of claim 90 or claim 91, wherein the solid surface is a bead.

97. The system of claim 83, wherein the paired fluorophore comprises BHQ1.

98. The system of claim 83, wherein the paired fluorophore complement comprises FAM.

99. The system of claim 83, wherein the paired fluorophore and the paired fluorophore complement comprise a FRET pair.

100. The system of claim 83, wherein the analyte does not comprise a nucleic acid.

101. The system of claim 83, wherein the analyte comprises a protein.

102. The system of claim 101, wherein the protein is a cell surface protein.

103. The system of claim 83, wherein the analyte comprises a viral particle.

104. The system of claim 83, wherein the analyte comprises an epitope.