Aptamer-capture agent-based molecular sensors and methods of use

Abstract

Provided are molecular sensors comprising a capture agent that binds to a first epitope of an analyte, and an aptamer that binds to a second epitope of the analyte. The aptamer is stably associated with the capture agent and produces a detectable signal upon cooperative binding of the capture agent and aptamer to the analyte. Also provided are substrates comprising a molecular sensor of the present disclosure immobilized on the surface thereof, as well as devices comprising the molecular sensors and substrates. Methods of using the molecular sensors, substrates and devices of the present disclosure are also provided. In some instances, the methods are for assessing a sample for an analyte, the methods comprising contacting a molecular sensor of the present disclosure with the sample under conditions suitable for cooperative binding of the capture agent and aptamer to the analyte, and assessing for a detectable signal produced by the aptamer.

Description

[0001] APTAMER-CAPTURE AGENT-BASED MOLECULAR SENSORS AND METHODS OF USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 529,633, filed July 28, 2023, which application is incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with Government support under contract OD025342 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] INTRODUCTION

[0007] Most approaches to molecular detection are designed for single-timepoint measurements, but there is considerable value in being able to track changes in analyte concentration dynamically. This is particularly important when tracking clinically-relevant markers in biofluids, where continuous measurements can provide a real-time indicator of disease states1 2, metabolic activity3, hormonal dysfunction45, and other physiological processes67. However, it remains a challenge to develop simple, generalizable biosensor architectures that are suitable for continuous molecular detection. Monoclonal antibodies generally offer the best sensitivity and specificity, but conventional immunoassays such as enzyme-linked immunosorbent assays (ELISA) require the use of a secondary reporter antibody, and thus are limited to single-timepoint measurements89. While one emerging approach has been shown to adapt antibodies into a molecular pendulum construct that achieves reversible binding-induced electrochemical signaling10, this approach relies only on the antibody’s intrinsic properties and thus has limited tunability to optimize its sensitivity. In contrast to antibodies, it is relatively straightforward to engineer and tune nucleic acid-based aptamers to undergo reversible conformation changes upon target. When coupled with a reporter moiety such as a redox tag or fluorophore, these aptamer-based molecular switches can be used to continuously measure target molecule concentrations directly in complex samples1 15. For example, the commonly used intramolecular strand displacement design (ISD), in which signaling is inhibited in the absence of target by a complementary DNA strand that hybridizes to the aptamer’s target-binding domain, offers a generalizable approach to aptamer switch development16,17. Nevertheless, aptamer switch design approaches introduce important trade-offs, as competition-based switching mechanisms can greatly reduce aptamer affinity18. Given that aptamers generally exhibit lower target affinity than antibodies, this can render them insufficiently sensitive for real-world detection applications. While there are alternative strategies to designing structure-switching aptamers such as screening or truncation, these strategies still provide aptamers with limited sensitivity, especially for protein biomarkers where physiological concentrations are often sub-nanomolar19. SUMMARY

[0008] Provided are molecular sensors comprising a capture agent that binds to a first epitope of an analyte, and an aptamer that binds to a second epitope of the analyte. The aptamer is stably associated with the capture agent and produces a detectable signal upon cooperative binding of the capture agent and aptamer to the analyte. Also provided are substrates comprising a molecular sensor of the present disclosure immobilized on the surface thereof, as well as devices comprising the molecular sensors and substrates. Methods of using the molecular sensors, substrates and devices of the present disclosure are also provided. In some instances, the methods are for assessing a sample for an analyte, the methods comprising contacting a molecular sensor of the present disclosure with the sample under conditions suitable for cooperative binding of the capture agent and aptamer to the analyte, and assessing for a detectable signal produced by the aptamer.

[0009] B IEF DESCRIPTION OF THE FIGU ES

[0010] FIG. 1 : Schematic of the intramolecular strand displacement (ISD) switch and programmable antibody and DNA aptamer switch (PANDAS) design concept, (a) For the ISD switch, an aptamer is linked to a partially complementary displacement strand (DS) via a poly-T linker, and the two ends of the DNA molecule are labeled with a fluorophore and quencher moiety. Target binding leads to release of the DS and physical separation of these two elements, resulting in increased fluorescence, (b) In the PANDAS design, the ISD is coupled to the Fc domain of an antibody by a 30-nt oligonucleotide linker. The antibody will generally have much higher affinity than the aptamer, capturing the free target first and bringing it into close proximity with the ISD, allowing the captured target molecule to bind the ISD and induce a conformational change that produces a fluorescent readout.

[0011] FIG. 2: (a) A simplified PANDAS model considers five conformations for the aptamer (Ap), antibody (Ab), and target (yellow square): unbound (U), target singly-bound to the aptamer (BAP) or antibody (BAD), the bivalently-bound conformation (BAp,Ab), and the doubly bound conformation (BApiB b). (b) Tuning of PANDAS affinity by changing ISD aptamer affinity. The plot shows the fraction of fluorescently active constructs at various concentrations of target in a scenario where KDAb = 3 nM, Ceft = 3 pM, and KD Pranges from 30 nM-30 pM. Solid lines represent plots of the full thermodynamic binding model. Dashed lines represent the simplified single-isotherm approximation used when KDAp« Ceff. O markers represent the dual-isotherm approximation used when KDAp-Ceff-

[0012] FIG. 3: (a) Testing the specificity of PANDAS cooperative binding. A flow-cytometry- based assay was used to measure the binding-induced fluorescent readout from the thrombin PANDAS in the presence of thrombin and BSA compared to a non-cooperative PANDAS construct with a mismatched anti-TNF-a antibody exposed to thrombin or TNF-a. (b) Tuning the binding affinity of the thrombin PANDAS construct. The length of the DS sequence in the PANDAS was tailored to range from 6-9 nt and then used flow cytometry to measure fluorescence after 30 min of incubation with varying concentrations of thrombin. Plot show fluorescence measurements from full PANDA constructs incorporating DS of varying lengths. All datapoints and error bars represent the mean and standard deviation of three replicates.

[0013] FIG. 4: PANDAS kinetics and reversibility of target binding, (a) To measure the kinetics of PANDAS_ds7, dissociation curves were generated by washing thrombin-saturated PANDAS beads with buffer or 1 pM thrombin and measuring fluorescence every three minutes, (b) To assess reversibility, PANDAS_ds7-coupled beads were transferred between buffer and 1 pM thrombin, collecting fluorescence measurements 15 minutes after each shift. As controls, fluorescence measurements were collected from PANDAS-ds7 beads that were maintained in either buffer or 1 pM thrombin, (c) PANDAS deployment in an interferent-rich environment. PANDAS_ds7 maintained consistent thrombin-binding performance in buffer alone or containing -530 pM albumin, (d) Also tested was the sensor’s capacity for continuous sensing of a physiological range of thrombin concentrations in buffer containing 530 pM albumin, with data collected every 15 min. Figure a, b and c datapoints and error bars represent the mean and standard deviation of three replicates.

[0014] FIG. 5: Assembly process for the PANDAS construct. An azide handle is attached to the Fc domain of a native antibody by performing galactosidase digestion followed by UDP-GalNAz addition. In parallel, a DBCO handle is attached to an amine-modified DNA anchor (‘Ab2 adapter’) using DBCO-NHS. This DNA anchor is then conjugated to the antibody’s azide group via a copper-free click reaction. This oligonucleotide-modified antibody can now be hybridized to the aptamer switch, yielding the final PANDAS construct.

[0015] FIG. 6: Assessing PANDAS antibody-aptamer compatibility. Antibody F1 was immobilized to protein G beads as first layer. Thrombin or water was added as second layer and fluorescent aptamer was added as third layer. Figure a represented F1 -thrombin-TBA. Figure b represented F1 -water- TBA. Figure c represented F1-thrombin-HD22. Figure d represented F1 - water-HD22. Figure a and b assessed antibody F1 and aptamer TBA compatibility in the presence (a) and absence (b) of thrombin. Figure c and d assessed antibody F1 and aptamer HD22 compatibility in the presence (c) and absence (d) of thrombin. A small shift was observed in F1 and TBA pairs.

[0016] FIG. 7: PANDAS antibody aptamer compatibility was assessed by constructing sandwich layers on Protein G beads. In these experiments, antibody 5020 was immobilized to protein G beads as first layer. Thrombin or water was added as second layer and fluorescent aptamer was added as third layer. Figure a represented 5020-thrombin-TBA. Figure b represented 5020-water- TBA. Figure c represented 5020-thrombin-HD22. Figure d represented 5020-water-HD22. Figure a and b assessed antibody 5020 and aptamer TBA compatibility in the presence (a) and absence (b) of thrombin. Figure c and d assessed antibody 5020 and aptamer HD22 compatibility in the presence (c) and absence (d) of thrombin. 5020 / TBA pair and 5020 / HD22 pair both produced significant fluorescent shifts.

[0017] FIG. 8: BLI affinity measurements of antibody 5020. AMC tips were pre-hydrated in ISD buffer for 20 minutes and then in 20 nM 5020 antibody to achieve 0.5 nm loading level. After ligand loading, sensors were dipped in 10, 20, 40, or 80 nM thrombin for 300 sec and then dipped in buffer for 600 sec. Signals were normalized by subtraction of the signal from reference tips. ForteBio Octet Data Analysis software was used to align and fit the data in 1 :1 binding mode (Kon= 6.09 x105M1s1, KOff =1 .68x103s1and KD=2.76x109M).

[0018] FIG. 9: BLI affinity measurements of aptamer TBA. SA tips were pre-hydrated in ISD buffer for 20 minutes and then in 20 nM TBA to achieve 0.5 nm loading level. After ligand loading, sensors were dipped in 25, 50, 100, or 200 nM thrombin for 300 sec and then dipped in buffer for 600 sec. Signals were normalized by subtraction of the signal from reference tips. ForteBio Octet Data Analysis software was used to align and fit the data in 1 :1 binding mode (Kon= 7.29x104M1s1, Koff =8.27x103s1and KD=1 .14x107M).

[0019] FIG. 10: BLI affinity measurements of the TBA-based ISD. SA tips were pre-hydrated in ISD buffer for 20 minutes and then in 20 nM TBA ISD to achieve 0.5 nm loading level. After ligand loading, sensors were dipped in 25, 50, 100, 200 nM thrombin for 300 sec and then dipped in buffer for 600 sec. Signals were normalized by subtraction of the signal from reference tips. ForteBio Octet Data Analysis software was used to align and fit the data in 1 :1 binding mode (Kon= 4.12x104M1s1, Kotf =1 ,01 x102s1and KD=2.46x107M).

[0020] FIG. 11 : PANDAS antibody labeling was assessed via 10% Nu-PAGE. Lane 1 was loaded with SeeBlue ladder. Lane 2 was loaded with unmodified antibody 5020. Lane 3 was loaded with modified 5020 in preparation for PANDAS assembly. In Lane 3, the shifted heavy chain and unmodified heavy chain are present at a roughly 1 :1 ratio, indicating that each mAb has one oligo adapter on average.

[0021] FIG. 12: Indirect and direct conjugation approaches that may be employed to produce molecular sensors of the present disclosure according to some embodiments.

[0022] FIG. 13: Intermolecular strand-displacement aptamer switch-based designs that may be employed in molecular sensors of the present disclosure according to some embodiments.

[0023] DETAILED DESCRIPTION

[0024] Before the molecular sensors and methods of the present disclosure are described in greater detail, it is to be understood that the molecular sensors and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the molecular sensors and methods will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the molecular sensors and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the molecular sensors and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the molecular sensors and methods.

[0025] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the molecular sensors and methods belong. Although any molecular sensors and methods similar or equivalent to those described herein can also be used in the practice or testing of the molecular sensors and methods, representative illustrative molecular sensors and methods are now described.

[0027] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present molecular sensors and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0028] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0029] It is appreciated that certain features of the molecular sensors and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the molecular sensors and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present molecular sensors and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0030] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0031] MOLECULAR SENSORS, SUBSTRATES AND DEVICES

[0032] Aspects of the present disclosure include molecular sensors. In certain embodiments, the molecular sensors comprising a capture agent that binds to a first epitope of an analyte, and an aptamer that binds to a second epitope of the analyte, where the aptamer is stably associated with the capture agent and produces a detectable signal upon cooperative binding of the capture agent and aptamer to the analyte. The capture agent mediates initial analyte binding due to its higher affinity, thereby increasing the local analyte concentration and leading to cooperative binding and signaling by the aptamer, e.g., a strand-displacement (SD) aptamer switch. As proof of concept and as detailed in the Experimental section below, a molecular sensor according to embodiments of the present disclosure exhibited a surprising enhancement in sensitivity as compared to the aptamer alone. Moreover, the molecular sensor exhibited reversible binding, enabling repeated measurements and retaining remarkable sensitivity even in interferent-rich samples. Details regarding embodiments of the molecular sensors will now be provided.

[0033] As summarized above, the molecular sensors of the present disclosure comprise a capture agent. A variety of suitable capture agents may be employed, non-limiting examples of which include a receptor (e.g., when the analyte is a ligand of the receptor), a ligand (e.g., when the analyte is a receptor for the ligand), a small molecule, an antibody, a T-cell receptor (TCR), etc.

[0034] For example, in some instances, the capture agent is an antibody. By “antibody” is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., lgG1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the analyte. In certain embodiments, the antibody is an IgG, Fab, scFv, scFv- Fc, scFv-CH3, scFv-zipper, scFab, VHH / VH, or diabody. The capture agent generally will exhibit specific binding to the analyte. The phrases “specifically binds”, “specific for”, “immunoreactive” and “immunoreactivity”, and “antigen binding specificity”, when referring to a capture agent (e.g., an antibody), refer to a binding reaction with the analyte which is highly preferential to the analyte or a fragment thereof, so as to be determinative of the presence of and / or selective for the analyte in the presence of a heterogeneous population of antigens (e.g., proteins and other biologies, e.g., in a sample or in vivo). Thus, under designated assay (e.g., immunoassay) conditions, the specified capture agent binds to the analyte and does not bind in a significant amount to other antigens present in the sample. In some embodiments, the capture agent “specifically binds” the analyte if it binds to or associates with the analyte with an affinity or Ka(that is, an equilibrium association constant of a particular binding interaction with units of 1 / M) of, for example, greater than or equal to about 105M1. In certain embodiments, the capture agent binds to the analyte with a Kagreater than or equal to about 106M1, 107M1, 108M1, 109M1, 1 O10M1, 1011M1, 1012M1, or 1013M1. “High affinity” binding refers to binding with a Kaof at least 107M1, at least 108M1, at least 109M1, at least 101° M1, at least 1011M1, at least 1012M1, at least 1013M1, or greater. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 105M to 1013M, or less). In some embodiments, specific binding means the capture agent binds to the analyte with a KDof less than or equal to about 105M, less than or equal to about 106M, less than or equal to about 107M, less than or equal to about 108M, or less than or equal to about 109M, 1010M, 1011M, or 1012M or less. The binding affinity of the capture agent for the analyte may be known, or can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0035] As used herein, an “aptamer” is a nucleic acid with low molecular weight (e.g., ranging from 5-40 kDa), which forms a specific three-dimensional structure and exhibits specific binding to the analyte. It will be appreciated upon review of the present disclosure that the aptamer may be present within an aptamer switch (e.g., comprising an intramolecular or intermolecular displacement strand), where the aptamer switch may be referred to as an “aptamer”. An aptamer may comprise deoxyribonucleotide monomers, ribonucleotide monomers, non-natural nucleotide monomers, or any combination thereof. In some instances, the aptamer comprises one or more non-natural nucleotides which increase the stability of the aptamer, e.g., thermostability, stability against enzymatic degradation, and / or the like, as compared to the aptamer lacking the one or more non-natural nucleotides. According to some embodiments, an aptamer comprises one or more thermostability-increasing nucleotides. Non-limiting examples of such nucleotides include 2-Amino-2'-deoxyadenosine-5'-Triphosphate (2-Amino-dATP), 5-Methyl-2'-deoxycytidine-5'- Triphosphate (5-Me-dCTP), 5-Propynyl-2'-deoxycytidine-5'-Triphosphate (5-Pr-dCTP), 5- Propynyl-2'-deoxyuridine-5'-Triphosphate (5-Pr-dUTP) and / or halogenated deoxy-uridine (Xdll) such as 5-Chloro-2'-deoxyuridine-5'-Triphosphate (5-CI-dUTP), 5-Bromo-2'-deoxyuridine-5'- Triphosphate (5-Br-dUTP), or any combination thereof. In certain embodiments, an aptamer comprises one or more internucleotide linkages resistant to nuclease degradation. For example, the aptamer may comprise a phosphorothioate linkage, a boranophosphate linkage, a phosphorodiamidate linkage, a phosphoamidate linkage, a thiophosphoramidate linkage, or any combination thereof. In some instances, the one or more internucleotide linkages resistant to nuclease degradation comprise one or more phosphorothioate linkages. According to some embodiments, the backbone of the aptamer comprises a mixture of phosphodiester and phosphorothioate linkages. In other embodiments, the entire backbone of the aptamer comprises phosphorothioate linkages.

[0036] A variety of suitable aptamers may be employed in the molecular sensors of the present disclosure. In certain embodiments, an aptamer switch is employed. An aptamer switch, in some embodiments, comprises an aptamer that binds the second epitope of the analyte and may comprise additional elements that act in combination with the aptamer, such that, upon analyte binding, a measurable signal is generated. In some embodiments, target binding induces a conformational change in the aptamer and / or elements of the aptamer switch to create a measurable signal.

[0037] In some implementations, the aptamer switch comprises a strand displacement aptamer switch, as known in the art. The strand displacement aptamer switch may comprise a nucleic acid duplex formed between a labeled target-binding aptamer and a labeled complementary strand, wherein, in the absence of target, the target-binding aptamer and complementary strand are in a hybridized state, producing no signal and wherein, upon target binding to the aptamer, the short complementary strand is displaced, changing the relative position of the two labels and producing a measurable signal.

[0038] In various implementations, the aptamer switch may comprise a split aptamer sensor, comprising a target-binding aptamer divided into two strands that, upon target binding assemble into a secondary structure that generates a detectable signal.

[0039] In some implementations, the aptamer switch comprises a pair of complementary fluorescent reporter moieties wherein the relative position of the moieties to each other modulates the signal generated thereby, for example a pair of moieties comprising a fluorophore and a quencher. Exemplary fluorophores include, for example, fluorescein, carboxyfluorescein, 6- carboxyfluorescein carboxytetramethylrhodamine, Cy-3, Cy-5, and quantum dots; and exemplary quenchers include DABCYL, BHQ-1 , and BHQ-2.

[0040] In another implementation, the aptamer switch comprises a turn-on aptamer switch. Implementations the turn-on aptamer switch comprises an aptamer functionalized with a conditional fluorophore, wherein binding to the target induces a change in conditions of the conditional fluorophore such that it generates a measurable signal. Exemplary conditional fluorophores include, for example, OTB-SO3, DFHBI, DFHO, DIR, DMHBO, and SR-DM.

[0041] Examples of suitable aptamer switches include, but are not limited to, stranddisplacement (SD)-based aptamer switches, e.g., with an intramolecular strand displacement (ISD) design, with an intermolecular strand displacement design (e.g., a duplex-bubble switch (DBS) design), or the like. For example, in some embodiments, the molecular sensor comprises an ISD-based aptamer switch in which the aptamer is linked to a displacement strand (DS) domain complementary to a binding motif of the aptamer that binds to the analyte. The DS domain is displaced upon cooperative binding of the capture agent and aptamer to the analyte. In other embodiments, the molecular sensor comprises an aptamer switch with an intermolecular strand displacement design (e.g., a duplex-bubble switch (DBS) design) in which the aptamer is hybridized to a domain of a displacement strand (DS) complementary to a binding motif of the aptamer that binds to the analyte, where the domain of the DS is displaced upon cooperative binding of the capture agent and aptamer to the analyte, and where the aptamer and DS are separate nucleic acid molecules. According to some embodiments, when a strand-displacement (SD)-based aptamer switch is employed, the detectable signal is produced upon displacement of the DS domain.

[0042] According to some embodiments, the detectable signal is an optically-detectable signal. For example, the detectable signal may be a fluorescent signal, e.g., where the aptamer or a displacement strand (if present) is labeled with a fluorophore. Non-limiting examples of approaches for the production of fluorescent signals upon cooperative binding of the capture agent and aptamer to the analyte include fluorophore-quencher interactions, Forster resonance energy transfer (FRET), and the like. In certain embodiments, the aptamer comprises a quencher moiety that quenches the fluorophore in a first conformation when the molecular sensor is not bound to the analyte, and upon cooperative binding of the capture agent and aptamer to the analyte, the aptamer assumes a second confirmation in which the fluorophore assumes an unquenched state.

[0043] In certain embodiments, the molecular sensors of the present disclosure are configured to produce a detectable signal other than an optically-detectable signal. In some instances, the detectable signal is an electrochemical signal. For example, the molecular sensors may undergo analyte binding-induced changes in electron-transfer kinetics of a redox reporter that can be monitored (e.g., in real time) via electrochemical interrogation. In some embodiments, an aptamer dually labeled with thiol and ferrocene groups is hybridized with a complementary strand (intramolecular or intermolecular), and the thiolated duplex is immobilized (e.g., self-assembled) on a gold electrode. The duplex is responsive to the analyte, which liberates the complementary strand and forms a complex with the analyte. The electroactive ferrocene moiety, which is distal to the electrode surface in the absence of the analyte, is moved to the proximal position during the binding-induced structural transition. This binding turns on the electron transfer and leads to measurable electrochemical signals for detection (e.g., quantification) of the analyte.

[0044] The terms “complementary” or “complementarity” as used herein refer to a nucleotide sequence of a first nucleic acid that base-pairs by non-covalent bonds to a region of a second nucleic acid, or a nucleotide sequence of a first region of a nucleic acid that base-pairs by non- covalent bonds to a second region of the nucleic acid, i.e., intramolecularly. In the canonical Watson-Crick-Franklin base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). As such, A is complementary to T and G is complementary to C. In RNA, A is complementary to U and vice versa. Typically, “complementary” or “complementarity” refers to a nucleotide sequence that is at least partially complementary. These terms may also encompass duplexes that are fully complementary such that every nucleotide in one strand is complementary to every nucleotide in the other strand in corresponding positions. In certain cases, a nucleotide sequence may be partially complementary to a target, in which not all nucleotides are complementary to every nucleotide in the target nucleic acid in all the corresponding positions. For example, a region of a first nucleic acid may be perfectly (i.e., 100%) complementary to a region of a second nucleic acid, or the region of the first nucleic acid may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%, 90%, 95%, 99%). The percent identity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment). The nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity= # of identical positions / total # of positionsxl OO). When a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, then the molecules are identical at that position. A non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In some embodiments, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., wordlength=5 or wordlength=20).

[0045] The aptamer is stably associated with the capture agent. As used herein, “stably associated” means a physical association between two entities in which the mean half-life of association is one day or more in PBS at 4°C. In some embodiments, the physical association between the two entities has a mean half-life of one day or more, one week or more, one month or more, including six months or more, e.g., 1 year or more, in PBS at 4°C. According to some embodiments, the stable association arises from a covalent bond between the two entities, a non-covalent bond between the two entities (e.g., an ionic or metallic bond), or other forms of chemical attraction, such as hydrogen bonding, Van der Waals forces, and the like.

[0046] In certain embodiments, the aptamer is stably associated with the capture agent through a covalent linkage. For example, the molecular sensor may be a capture agent-aptamer conjugate. Bioconjugation strategies that find use in conjugating the aptamer (or aptamer switch) and capture agent are described in Hermanson, “Bioconjugate Techniques,” Academic Press, 3rd edition, 2013, Haugland, 1995, Methods Mol. Biol. 45:205-21 ; Brinkley, 1992, Bioconjugate Chemistry 3:2, and elsewhere. For example, the capture agent may be functionalized (or “activated” / “derivatized”) with reactive groups to which a linker or aptamer / aptamer switch may bind for conjugation to the aptamer / aptamer switch, or vice versa.

[0047] Functional groups that may be used to bind components of the molecular sensors of the present disclosure include, but are not limited to, active esters, isocyanates, imidoesters, hydrazides, amino groups, aldehydes, ketones, photoreactive groups, maleimide groups, alpha- halo-acetyl groups, epoxides, azirdines, and the like. Reagents such as iodoacetamides, maleimides, benzylic halides and bromomethylketones react by S-alkylation of thiols to generate stable thioether products. For example, at pH 6.5-7.5, maleimide groups react with sulfhydryl groups to form stable thioether bonds. Arylating reagents such as NBD halides react with thiols or amines by a similar substitution of the aromatic halide by the nucleophile. Because the thiolate anion is a better nucleophile than the neutral thiol, cysteine is more reactive above its pKa(~8.3, depending on protein structural context). Thiols also react with certain amine-reactive reagents, including isothiocyanates and succinimidyl esters. The TS-Link series of reagents are available for reversible thiol modification.

[0048] With respect to amine reactive groups, primary amines exist at the N-terminus of polypeptide chains and in the side-chain of lysine (Lys, K) amino acid residues. Among the available functional groups in typical biological or protein samples, primary amines are especially nucleophilic, making them ready targets for conjugation with several reactive groups. For example, NHS esters are reactive groups formed by carbodiimide-activation of carboxylate molecules. NHS ester-activated crosslinkers and labeling compounds react with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (NHS). Also by way of example, imidoester crosslinkers react with primary amines to form amidine bonds. Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half-life and reactivity with amines increases. As such, crosslinking is more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine formation is favored between pH 8-10.

[0049] Numerous other synthetic chemical groups will form chemical bonds with primary amines, including but not limited to, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, carbodiimides, anhydrides, and fluorophenyl esters. Such groups conjugate to amines by either acylation or alkylation.

[0050] Approaches for chemical attachment of the capture agent and aptamer / aptamer switch also include, but are not limited to, click-chemistry based approaches. Click-chemistry reactions that may be employed include (i) nucleophilic substitutions; (ii) additions to C-C multiple bonds (e.g., Michael addition, epoxidation, dihydroxylation, aziridination); (iii) nonaldol like chemistry (e.g., N-hydroxysuccinimide active ester couplings); and (iv) cycloadditions (e.g., Diels-Adler reaction, Huisgen’s cycloaddition). Huisgen’s cycloaddition has been applied in various branches of chemistry. It consists of the condensation of organic azides with alkyne groups to form 1 ,2,3- triazole linkages. Azide and alkyne functionalities can be easily introduced in the scaffold of large organic constructs of biological relevance. The reaction may be catalyzed by introducing copper(l). The Cu(l) core has a dual effect in that it activates the slow-reacting alkyne group thus accelerating the azide-alkyne condensation kinetics by — 107— 108-fold, and it organizes the reacting groups by “templation” so that only a regiospecific 1 ,4-disubstituted adduct is formed. This reaction is known as the copper-catalyzed azide alkyne cycloaddition (CuAAC), and its compatibility with a wide range of biological substrates and synthetic conditions makes CuAAC the flagship among click conjugations. Since its discovery, Cu(l)-catalyzed azide alkyne cycloaddition has been widely used within the fields of biology, biochemistry, and biotechnology. Click-chemistry reactions that may be employed to attach the nucleotide or polynucleotide to the particle include, but are not limited to, Huisgen Azide-Alkyne 1 ,3-Dipolar Cycloaddition, Copper- Catalyzed Azide-Alkyne Cycloaddition (CuAAC), Ruthenium-Catalyzed Azide-Alkyne Cycloaddition (RuAAC), and the like. Details regarding click-chemistry with nucleic acids are found, e.g., in Fantoni et al. (2021 ) Chem. Rev. 121 (12):7122— 7154.

[0051] According to some embodiments, the capture agent comprises a polypeptide site- specifically conjugated to the aptamer. In one non-limiting example, the capture agent is an antibody comprising a constant region, and the aptamer is site-specifically conjugated to the constant region. In some instances, the antibody comprising an Fc region and the aptamer is site-specifically conjugated to the Fc region. Particular non-limiting approaches for conjugation of the aptamer to a proteinaceous capture agent (e.g., an antibody, protein ligand, protein receptor, or the like) will now be described in the context of antibody-aptamer conjugates.

[0052] An aptamer, displacement strand, or linker can be covalently and site-specifically linked to the glycan moieties within the antibody Fc region using enzymatic methods to site-specifically introducing azide-functionalized monosaccharides within these glycans, followed by clickchemistry reaction of the introduced azide groups with DNA that is functionalized with partner click handles such as Dibenzocyclooctyne (DBCO) or alkynes. This enzymatic labeling method is decribed in: www.ncbi.nlm.nih.gov / pmc / articles / PMC9154768 (relevant vendor of kit: thermofisher.com / order / catalog / product / S10900). An aptamer, displacement strand, or linker can be covalently and site-specifically linked to the antibody Fc region by first crosslinking the antibody to a protein G-derived reagent that forms a covalent bond with the antibody upon light exposure. This crosslinking protein can then contain a secondary chemical handle, such as a thiol, azide, DBCO, or tetrazine, which can be subsequently linked to the aptamer, displacement strand, or linker through a specific reaction with a maleimide, alkyne / DBCO, azide, or trans-cyclooctene (TOO) respectively. This labeling method is described in: pubs. acs.org / doi / 10.1021 / acs.bioconjchem.5b00275 (relevant vendor of kit: alphathera.com / ).

[0053] An aptamer, displacement strand, or linker can be covalently and site-specifically linked to the interchain disulfide bonds of the antibody by use of a primary reagent that reacts with both cysteine residues of a reduced disulfide bond, rebridging the disulfide bond. This reagent can then contain a secondary chemical handle for conjugation to a modified DNA oligo (possible reactive pairs described above. An example of this method is described in: pubs. acs.org / doi / 10.1021 / bc500148x.

[0054] An aptamer, displacement strand, or linker can be non-specifically and stochastically conjugated to either lysine or cysteine residues within the antibody structure by reaction with DNA modified with amine reactive (es. carboxylic acid esters) or thiol reactive (maleimides or iodoacetamides) groups respectively. This non-specific conjugation can also be achieved by first reacting the antibody with an amine or thiol reactive probe that contains a secondary reactive handle (list provide above), followed by conjugation with DNA bearing the reactive partner to that handle. An example of this method is described in: nature.com / articles / s41598-020-58238-6.

[0055] An aptamer, displacement strand, or linker can be covalently and site-specifically linked to defined positions within the antibody structure by expressing antibodies that contain unnatural amino acids with orthogonal chemical reactivity, followed by orthogonal conjugation with chemically modified DNA. An example of this approach is presented in: pnas.org / doi / abs / 10.1073 / pnas.1211023109.

[0056] As noted above, the linkage between the aptamer and capture agent (e.g., antibody) can take the form of either direct conjugation of the aptamer to the capture agent, or hybridization of part of the aptamer sequence to an anchor DNA sequence conjugated to the capture agent. As such, the linker region may be composed of a combination of regions of the anchor strand and the aptamer strand. These two examples are illustrated in FIG. 12.

[0057] In the anchor hybridization embodiment, the minimum linker length will be dictated by the length required for stable hybridization between the anchor and aptamer sequence. The shortest possible length would be 10 bp for stability, and in this shortest case the 10 bp linker would be double stranded. In some embodiments, the longest reasonable linker length would be 60-80 nucleotides total, and this length could include regions that are double stranded (including anchor-aptamer hybridization, or hybridization of third strands to the linker sequence) or single stranded, as well as including mismatches, nicks, or bulges. The sequence base composition could vary between 100% A / T to 100% G / C, although more A / T rich linkers may be advantageous. The linker could also include non-natural nucleobases, or include non-canonical DNA structures such as DNA triplexes, g-quadruplexes, or double crossover DNA associates (DX tiles, example: ncbi.nlm.nih.gov / pmc / articles / PMC3221779 / ).

[0058] In the direct conjugation case, the linker could be as short as 0 nt, and linkers in the range of 0-1 Ont could be made single- or double-stranded. In some embodiments, a maximum reasonable linker length would be 60-80 nucleotides total and could include the full design space described above.

[0059] The linker length may set the distance between the capture agent (e.g., antibody) and aptamer, positioning them with the desired proximity to their respective binding epitopes such that they can bind with the desired degree of cooperativity. An “epitope” is the small site on the analyte to which the capture moiety or aptamer may specifically bind, where the epitope may be a linear epitope or a conformational epitope. Single- vs double-stranded linkers (as well as triplexes, DX tiles) can modulate the linker rigidity such that cooperativity can be modulated, and different sizes of targets can be accommodated.

[0060] With the benefit of the present disclosure, capture agent-aptamer pairs may be developed using a variety of approaches. Non-limiting approaches in which the capture agent is referred to as an antibody will now be described.

[0061] A process for developing an antibody / aptamer pair suitable for this design may follow two steps: first, development of a structure switching aptamer with fluorescent target sensing capabilities or library of structure switching aptamers with target sensing capabilities, followed by determination of a suitable antibody / aptamer pair for recognition of separate epitopes through combinatorial screening of either multiple antibody candidates or multiple aptamer candidates.

[0062] Structure switching aptamer development: This process may begin by selecting for or designing an aptamer switch capable of fluorescently sensing the target, even with low affinity, so that it can be implemented within this design. First, a target-binding aptamer can either be selected through a range of existing aptamer selection techniques (see, e.g., US20210324374A1 and US10036008B2, the disclosures of which are incorporated herein by reference in their entireties for all purposes), or taken from existing literature or previous selection results. This target-binding aptamer can then be engineered to achieve switching and signaling by augmenting it with a linker or displacement strand domain that leads to fluorescent signaling upon target binding (see, e.g., US20210355496A1 , the disclosure of which is incorporated herein by reference in its entirety for all purposes). This could engineering also be achieved by high- throughput screening of auxiliary domains that introduce switching behavior into the aptamer. See, e.g., nature.com / articles / s41467-023-38105-4. Screening for antibody / aptamer pairs: To identify antibody pairs that cooperate with aptamer binding by recognizing a separate epitope, aptamers that bind and signal in response to target binding can then be screened for their signaling in response to the same target that is prebound with candidate antibodies. If the signaling ability is maintained, this suggests that the antibody candidate does not interfere with the same epitope, and thus offers a suitable pair. This can be performed with a fixed candidate aptamer and a range of candidate antibodies. Alternatively, if only a single candidate antibody is available, this same complementary binding behavior can be assessed by measuring the binding response of a range of candidate aptamers when exposed to target pre-bound with the antibody candidate. Successful pairs with noninterfering binding could then be further screened to ensure sandwich binding using bead-based assays (See Experimental section and FIG. 6). These successful pairs could then be stably associated to produce a molecular sensor of the present disclosure.

[0063] As noted above, a range of aptamer-switch designs that use two hybridized strands (each strand containing a subset of the domains needed for switching) can be implemented. A nonlimiting example of such a design is schematically illustrated in FIG. 13.

[0064] Aspects of the present disclosure further include substrates. In some aspects, provided are substrates comprising any of the molecular sensors of the present disclosure immobilized on the surface thereof. Such substrates may comprise one or a plurality of the molecular sensors.

[0065] In certain embodiments, the substrate is comprised within a well or channel. In some instances, the substrate is a hydrogel, a microneedle or an optical fiber.

[0066] According to some embodiments, the substrates are microparticles. By “microparticles” is meant a collection of solid supports having an average greatest dimension of 1000 micrometers (pm) or less. In some embodiments, the collection of solid supports has an average greatest dimension of 750 pm or less, 500 pm or less, 250 pm or less, 100 pm or less, 1 pm or less, 0.75 pm or less, 0.50 pm or less, 0.25 pm or less, or 0.1 pm or less. In certain embodiments, the solid supports have an average greatest dimension of from about 0.50 pm to about 500 pm, e.g., from about 0.75 pm to about 250 pm, e.g., about 1 pm.

[0067] A variety of materials can be used as the solid supports. Support materials include any material that can act as a support for attachment of the molecular sensors. Suitable materials include, but are not limited to, organic or inorganic polymers, natural and synthetic polymers, including, but not limited to, agarose, cellulose, nitrocellulose, cellulose acetate, other cellulose derivatives, dextran, dextran-derivatives and dextran co-polymers, other polysaccharides, glass, silica gels, gelatin, polyvinyl pyrrolidone, rayon, nylon, polyethylene, polypropylene, polybutylene, polycarbonate, polyesters, polyamides, vinyl polymers, polyvinylalcohols, polystyrene and polystyrene copolymers, polystyrene cross-linked with divinylbenzene or the like, acrylic resins, acrylates and acrylic acids, acrylamides, polyacrylamides, polyacrylamide blends, co-polymers of vinyl and acrylamide, methacrylates, methacrylate derivatives and co-polymers, other polymers and co-polymers with various functional groups, latex, butyl rubber and other synthetic rubbers, silicon, glass, and any combinations thereof.

[0068] The microparticles may be any suitable shape, including but not limited to spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped, cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped, nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped, prism-shaped, or any other suitable geometric or non-geometric shape.

[0069] In certain embodiments, the microparticles are beads. As used herein, the term “bead” refers to a small mass that is generally spherical or spheroid in shape. According to some embodiments, a bead as used herein has an average diameter of from about 0.50 pm to about 500 pm, e.g., from about 0.75 pm to about 250 pm, e.g., about 1 pm.

[0070] Also provided by the present disclosure are devices for analyte sensing comprising any of the molecular sensors or substrates of the present disclosure. In certain embodiments, a device of the present disclosure is a microfluidic or nanofluidic device. A microfluidic or nanofluidic device typically consists of components such as valves, pumps and mixers for manipulating and transporting the fluid at this scale. Various materials can be processed into miniaturized chips containing channels and chambers in the microscale range. A diverse repertoire of methods can be chosen to manufacture such platforms of desired size, shape, and geometry. Various kinds of materials can be used for the manufacturing of microfluidic devices. Typical substrates include glass, silicon, metals, polymers, and ceramics, but the diversity and quality of materials are continuously increasing.

[0071] In certain embodiments, a device of the present disclosure is a lateral flow device. The lateral flow assay (LFA) is a paper-based platform for the detection and quantification of analytes in complex mixtures, where the sample is placed on a test device and the results are displayed typically within 5-30 min. Lateral flow device formats which can incorporate the molecular sensors of the present disclosure for analyte sensing include those described in, e.g., Koczula & Gallotta (2016) Essays in Biochemistry 60(1 ):11 1 -120.

[0072] According to some embodiments, a device of the present disclosure is a test strip. Test strips for analyte detection and quantitation typically include a membrane test pad that is affixed to a solid support. The support may be a plastic (e.g., polystyrene, nylon, or polyester) or metallic sheet or any other suitable material. Associated with the test pad, e.g., coated onto the test pad, incorporated into the test pad, etc., may be a reagent composition comprising a plurality of molecular sensors of the present disclosure. The test strip may also be configured in more complex arrangements, e.g., where the test pad is present between the support and a surface layer, where one or more reagents employed in sample processing may be present on the surface layer. In addition, flow paths or channels may be present on the test strip.

[0073] In some instances, a device of the present disclosure is a microneedle patch. Wearable patches based on microneedle (MN) sensors provide minimally invasive analysis of analytes, typically through the interstitial fluid (ISF). Microneedle patch formats which can incorporate the molecular sensors of the present disclosure for analyte sensing include those described in, e.g., Lu et al. (2022) Front. Bioeng. Biotechnol. 10:851 134.

[0074] METHODS OF USE

[0075] As summarized above, aspects of the present disclosure further include methods of using the molecular sensors and devices of the present disclosure.

[0076] In certain embodiments, provided are methods of assessing a sample for an analyte. The methods comprise contacting any of the molecular sensors of the present disclosure with the sample under conditions suitable for cooperative binding of the capture agent and aptamer to the analyte. Such methods further comprise assessing for a detectable signal produced by the aptamer. In some instances, the contacting comprises contacting any of the substrates of the present disclosure with the sample.

[0077] The methods of the present disclosure find use in assessing a variety of samples for the analyte. In certain embodiments, the sample is a biological sample obtained from a subject. Biological samples of interest include, but are not limited to, a biological fluid. According to some embodiments, the biological fluid is blood or a fraction thereof (e.g., serum or plasma), urine, saliva, interstitial fluid (ISF), cerebrospinal fluid, tears, mucus, or sputum.

[0078] In certain embodiments, the analyte is a biomarker. Examples of biomarkers for which the sample may be assessed include protein biomarkers, small molecule biomarkers, lipid biomarkers, carbohydrate biomarkers, metabolite biomarkers, and the like. In some instances, the biomarker is a disease biomarker, e.g., a cancer biomarker, a cardiovascular disease biomarker, a neurological (e.g., neurodegenerative) disease biomarker, and the like. According to some embodiments, the biomarker is glucose or a ketone.

[0079] According to some embodiments, the biomarker is a therapeutic agent or metabolite thereof. For example, the methods find use in monitoring the presence or levels of a therapeutic agent in one or more samples obtained from a subject to whom the therapeutic agent is being administered. As used herein, a “therapeutic agent” is a physiologically or pharmacologically active substance that can produce a desired biological effect in a targeted site in an animal, such as a mammal or in a human. The therapeutic agent may be any inorganic or organic compound. A therapeutic agent may decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of disease, disorder, or cell growth in an animal such as a mammal or human. Examples include, without limitation, peptides, proteins, nucleic acids (including siRNA, miRNA and DNA), polymers, and small molecules.

[0080] In some instances, the analyte is a pathogen. Non-limiting examples of a pathogen for which the sample may be assessed include a virus, a bacterium, a fungus, a protozoan, or a worm. When the pathogen is a virus, the virus may be a coronavirus (e.g., a severe acute respiratory syndrome (SARS) coronavirus (e.g., (SARS-CoV-2)), an influenza virus, a Human herpesvirus (HHV, e.g., HHV-6), a herpes simplex virus (HSV, e.g., HSV-1 or HSV-2), or the like. When the pathogen is a bacterium, the bacterium may be Streptococcus, Staphylococcus, E. coli, or the like.

[0081] In certain embodiments, the analyte is a drug of abuse. Drugs of abuse include, but are not limited to, United States Drug Enforcement Administration (DEA) schedule I, schedule II, schedule III, schedule IV, and schedule V drugs. In certain embodiments, the one or more drugs of abuse comprises one or more of a narcotic, an opioid, a stimulant, a depressant, a hallucinogen, a cannabinoid, or any combination thereof. According to some embodiments, the one or more drugs of abuse comprises one or more opioids. In certain embodiments, the one or more opioids comprises oxycodone, hydrocodone, morphine, methadone, fentanyl, heroin, or any combination thereof. According to some embodiments, the one or more drugs of abuse comprises gabapentin.

[0082] According to some embodiments, the analyte is a performance enhancing drug. For example, the methods may include assessing the sample for one or more performance enhancing drugs selected from an anabolic steroid, androstenedione, human growth hormone (HGH), erythropoietin (EPO), a diuretic, creatine, a stimulant, or any combination thereof. In certain embodiments, when the methods comprise assessing the sample for one or more performance enhancing drugs, the subject from whom the sample was obtained is an upcoming, current, or former participant in an athletic contest.

[0083] The molecular sensor is contacted with the sample under conditions suitable for cooperative binding of the capture agent and aptamer to the analyte. Non-limiting examples of such conditions are provided in the Experimental section below. It will be appreciated that such conditions are generally those in which the capture agent and aptamer retain their three- dimensional structures, and those consistent with the ability of the capture agent and aptamer to bind their respective epitopes on the analyte. Conditions consistent with specific capture agentanalyte binding (e.g., antibody-antigen binding) and aptamer-analyte binding are known, and if useful may be readily optimized once a particular capture agent-aptamer pair has been selected for a particular analyte.

[0084] The assessment may be performed a single time on a single sample, multiple times on a single sample, or single or multiple times on multiple iterative samples, e.g., iterative biological samples obtained from a subject. For example, the methods may comprise assessing for the analyte at two or more time points, e.g., on the same sample or two iterative samples.

[0085] In certain embodiments, the methods are implemented using any of the devices of the present disclosure. For example, the methods may comprise a microfluidic assay, lateral flow assay, test strip-based assay, microneedle-based assay, or microneedle patch-based assay which employ the microfluidic devices, lateral flow devices, test trips, microneedles, or microneedle patches of the present disclosure. Such devices may comprise the components required for assessing for a detectable signal produced by the aptamer, which components may vary and will depend upon the detection method of choice, e.g., fluorescence detection, electrochemical detection, or any other convenient detection method which may be implemented using the molecular sensors of the present disclosure.

[0086] KITS

[0087] Also provided by the present disclosure are kits. In some embodiments, the kits provide one or components that find use in performing any of the methods of the present disclosure.

[0088] In certain embodiments, provided are kits that comprise any of the molecular sensors of the present disclosure, and instructions for using the molecular sensor to assess a sample for an analyte. According to some embodiments, the molecular sensors are provided in the form of a substrate or device of the present disclosure. For example, a kit may include microparticles (e.g., beads), a hydrogel, a microneedle, an optical fiber, or the like, comprising the molecular sensors immobilized on the surface thereof. Also by way of example, a kit may include any of the microfluidic devices, lateral flow devices, test strips, microneedle patches, etc. of the present disclosure. The kits may further comprise any components in addition to the molecular sensors (or substrates or devices) useful for carrying out the methods of the present disclosure.

[0089] Components of the kits may be present in separate containers, or multiple components may be present in a single container.

[0090] The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, Hard Disk Drive (HDD) etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0091] The following examples are offered by way of illustration and not by way of limitation.

[0092] EXPERIMENTAL

[0093] Presented in the work below is a molecular switch design that leverages both the high affinity of antibodies and the capacity to readily introduce intrinsic molecular switch functionality into aptamers. The non-limiting molecular switch design described in the Examples herein (sometimes referred to herein as Programmable ANtibody and DNA Aptamer Switch (or “PANDAS”)) links a structure-switching aptamer to the Fc-region of a modified antibody through a short DNA scaffold. Signaling is achieved through an ISD aptamer16 17design with a fluorescent readout. Normally, such sensors exhibit modest target affinity, but in the context of PANDAS, the affinity of the ISD aptamer is greatly enhanced through the effects of antibody-mediated cooperative binding. When the antibody binds the target molecule, the coupled ISD aptamer experiences a high local target concentration that promotes rapid binding and conformation change to generate a fluorescent signal.

[0094] As proof of concept, a PANDAS-based sensor was generated for the blood clotting protein thrombin. Demonstrated herein is that this construct can achieve ~1 OO-fold enhanced thrombin affinity relative to the aptamer switch on its own. Importantly, the ISD design also confers the ability to tune sensor affinity by altering the length of the displacement strand, and it is shown herein that an optimally-tuned version of this PANDAS can achieve an effective equilibrium dissociation constant (KD) of -100 pM with minimal background. Furthermore, this PANDAS construct binds reversibly, enabling continuous measurements of changing thrombin concentrations with ~10-minute temporal resolution to be obtained. The sensor design maintains excellent target specificity, and exhibited robust sensitivity and temporal resolution even when tested in an interferent protein-rich sample matrix. Given the modular design of the PANDAS construct, which requires no meaningful engineering of either the antibody or aptamer sequence beyond the introduction of a linker domain and displacement strand sequence, this strategy is expected to be broadly applicable for repurposing low-affinity aptamer switches to detect low- abundance targets in complex samples.

[0095] Example 1 - Rationale and design for an antibody-enhanced aptamer switch

[0096] The PANDAS reagent design takes advantage of the benefits of both aptamer switches and monoclonal antibodies. The aptamer component of the PANDAS construct makes use of the ISD design to achieve fluorescent signaling upon aptamer-target binding (Fig. 1a). Here, the aptamer is linked to a short displacement strand (DS) domain that is complementary to a portion of the aptamer’s binding motif. The DS hybridizes with the aptamer in the absence of target but is displaced by ligand binding as the free target concentration increases. This leads to targetdependent conformational switching of the ISD aptamer that can be fluorescently measured using a fluorophore-quencher pair coupled to the ends of the construct. However, because the ISD design introduces an intramolecular competitor within the aptamer switch that interferes with binding, the resulting ISD construct will always exhibit reduced affinity and temporal resolution compared to the native aptamer. The PANDAS design compensates for this reduced affinity by coupling the ISD aptamer switch to an antibody that recognizes the same target through a different epitope with higher affinity (see Fig. 5 for assembly process). The result is a hybrid molecular sensor that achieves high sensitivity through cooperative binding (Fig. 1 b). The high- affinity antibody binds the target at low concentrations at which the aptamer switch would not otherwise bind, and the resulting proximity between the aptamer and antibody-bound target induces aptamer binding, switching, and signaling at substantially lower concentrations than can be detected with the ISD alone. The ISD design also allows fine-tuning of the aptamer switch function through changes in its DNA sequence. For example, increasing the length of the DS will shift the equilibrium toward the fully folded and quenched state by forming a more stable stem structure17. This results in background signal suppression at the expense of decreased aptamer switch affinity and temporal resolution. Conversely, decreasing the DS length reduces the strength of intramolecular binding competition, thus increasing aptamer switch affinity and temporal resolution at the cost of a higher background signal.

[0097] The antibody component is site-specifically modified with a 30-nt DNA anchor at its Fc domain, which is in turn tethered to the aptamer switch component via hybridization to a complementary linker sequence that is attached to the fluorophore-tagged DS end of the ISD aptamer (all sequences shown in the Sequence Table below). This site-specific tethering ensures reproducible interactions between the target-binding sites of the aptamer and antibody, thereby enabling cooperative binding and reliable aptamer signaling. The modularity of this design allows for the ready substitution of different aptamer switches or antibody candidates within the PANDAS construct, enabling rapid testing of different switch configurations.

[0098] Sequence Table

[0099] Example 2 - Thermodynamic modeling of the PANDAS binding mechanism

[0100] To understand the degree of aptamer affinity enhancement that could be achieved, a thermodynamic model of target-induced binding and switching was developed for the PANDAS construct. The construct was modeled as two linked receptors: the aptamer and the antibody. The binding and switching of the chimeric construct are modeled in terms of target-dependent equilibria between five states, which in turn represent different combinations of target binding for the two receptors (Fig. 2a). In the majority of cases, the antibody affinity will be higher than that of the ISD aptamer, such that the antibody will capture the target first. The effective concentration of target in the proximity of the ISD aptamer will be dramatically increased due to the aptamerantibody linkage, and this high effective concentration will trigger aptamer binding and fluorescent signaling. It is assumed that the switch produces fluorescent signal only in states where the aptamer is bound to the target. In this model, the function of the PANDAS construct is dictated by three properties of the switch: KDAp, KDAband Ceff. Ceffis the effective concentration of target experienced by the aptamer when the target is already bound to the antibody, while KDApand KDAb are the equilibrium dissociation constants of the aptamer and antibody, respectively. While KDAband Ceffare not easily tunable within the PANDAS design, the abovementioned ISD tuning strategy allows us to control KDAp.

[0101] This model was leveraged to predict the binding response of the PANDAS construct for different values of KDAp(Fig. 2b; see Example 5 for details). Two different binding regimes for the PANDAS construct were identifed. The ‘antibody-assisted’ regime corresponds to values of

[0102] KDApwhere Ceff» KDAp» KDAb. In this case, once the target molecule is bound to the antibody, the aptamer readily binds it as well. As such, the aptamer-bound-only state, BAp, does not contribute meaningfully to the binding signal, and the fraction of fluorescent constructs is well- approximated by a single isotherm:

[0103] This approximation is shown as dashed lines in Fig. 2b. In the antibody-assisted regime, the PANDAS effective affinity is given by KDEff= KDAbKDAp / 2Cef), and thus depends linearly on the affinity of the ISD aptamer component, scaled by an enhancement factor of KDAb / (2Ceff). As KDApapproaches or exceeds Ceff(KDAp> Ceff» KDAb), the BAPstate becomes more prominent, as not all instances of target binding to the aptamer are necessarily triggered by an initial antibody binding event. In this ‘antibody-independent’ regime, the fraction of fluorescent PANDAS constructs is better approximated by a dual-isotherm given by:

[0104] This approximation is shown in Fig. 3b as O symbols. The first, high-affinity isotherm describes the antibody-assisted signal, while the latter, low-affinity isotherm describes the contribution of the BAP state. When KDAp» Ceff(i.e., aptamer affinity is so low that the proximity of antibody-bound targets is insufficient to trigger aptamer binding), the PANDAS binding response depends only on the affinity of the aptamer. As such, aptamers with extremely low affinities (e.g., in the mM range) will not experience sensitivity enhancement from a tethered high- affinity antibody, as the local enhancement of target concentration from proximity will be insufficient to trigger aptamer binding. Fortunately, most protein-binding aptamers exhibit affinities in the sub-micromolar range and are thus suitable for use in the PANDAS design.

[0105] Example 3 - Testing the PANDAS construct with a thrombin model system

[0106] As a proof-of-concept demonstration of the affinity enhancement that can be achieved with PANDAS, a fluorescent sensor for thrombin was designed. Thrombin is an important factor in the blood clotting process for which several well-characterized aptamers and monoclonal antibodies have been described20 25. Using flow cytometry, pairwise screening of potential aptamer (TBA, HD22) and antibody candidates (5020, F1 ) was performed to determine an appropriate aptamer-antibody pair. It was determined that the combination of the 5020 antibody and TBA aptamer achieved the most robust sandwich binding of thrombin (Fig. 6, 7).

[0107] The TBA sequence was then converted into an ISD-based fluorescent molecular switch. Initially, a DS length of 7 nucleotides was selected to achieve a high-amplitude switching signal with moderate binding affinity. Since the PANDAS enhancement mechanism depends on the affinity differences between the aptamer and antibody, biolayer interferometry (BLI) experiments were performed to determine the affinities of the individual switch components (Fig. 8-10). As expected, the TBA-based ISD aptamer switch exhibited lower affinity (KD = 246 nM) compared to the parent aptamer (KD= 1 14 nM), whereas the 5020 antibody had a much higher affinity (KD= 2.76 nM) than the aptamer switch. These results supported the suitability of this affinity reagent pair to achieve cooperative enhancement of aptamer binding at low target concentrations based on the model. 5020 antibodies were then site-specifically modified at their Fc region with a 30-nt anchor oligo adapter at a 1 :1 ratio (Fig. 11). Subsequently, the TBA ISD aptamer linker sequence was hybridized to this anchor oligo to produce the complete PANDAS construct. The 35-nt linker strand was selected to be sufficiently long to achieve robust antibody-aptamer hybridization with minimal dissociation, while not being so long that steric interference might impede sandwich binding. The assembled PANDAS construct was then immobilized onto microbeads coated with protein G, which binds to the antibody Fc region. These PANDAS-conjugated beads were incubated for 30 min in buffer spiked with a range of thrombin concentrations, followed by measurement of fluorescent switching using a flow cytometer. As a control, a non-cooperative PANDAS construct was generated in which the TBA ISD was coupled to an anti-TNFa antibody that does not bind the thrombin target and challenged this construct with the same range of thrombin concentrations. This assay showed that the PANDAS construct had a KD of ~1.7 nM (Fig. 3), achieving nearly 100-fold greater sensitivity relative to the non-cooperative control construct (KD -139.3 nM). That the switching mechanism was induced only by aptamer-specific binding was further validated. Non-specific effects of protein non-specific binding were ruled out by testing the anti-thrombin-based PANDAS construct against bovine serum albumin (BSA), as well effects of antibody binding without specific aptamer binding by challenging the anti-TNFa- based PANDAS construct with various concentrations of TNFa, and in both cases no fluorescent signal was observed. These experiments confirmed that signal generation was dependent on aptamer recognition of the target, whereas the affinity increase was contingent upon the specific and cooperative binding of both antibody and aptamer.

[0108] Example 4 - Rational sequence-based tuning of the PANDAS switch

[0109] Building off insights from the thermodynamic model, a range of PANDAS switches were developed in which the ISD design was tuned by extending or shortening the DS. It was predicted that elongating the DS would stabilize its hybridization to the aptamer and increase its competition with target binding, thereby decreasing the thrombin affinity of both the aptamer and the overall PANDAS construct. Conversely, shortening the DS should weaken intramolecular competition, leading to higher thrombin affinity for the aptamer and PANDAS. DS lengths ranging from 6-9 nt (using the naming convention Aptamer_ds#) were selected, the constructs were immobilized onto beads, and their thrombin binding affinity was quantified using the flow cytometry-based approach described above.

[0110] As expected, decreasing the DS length increased ISD aptamer binding affinity and thus shifted the dynamic range towards more accurate sensing of lower target concentrations. For example, decreasing the DS length from 7 to 6 nt led to a 15-fold decrease in Ko from 139.3 nM to 9.01 nM. Conversely, extending the DS length from 7 to 9 nt weakened the aptamer’s sensitivity considerably, yielding binding curves for which it proved difficult to achieve saturation. These ISD constructs were next assembled into full PANDAS constructs with the anti-thrombin antibody, and again their binding was assessed via flow cytometry (Fig. 3b). Between PANDAS_ds6 and PANDAS_ds9, a range of effective affinities spanning from 0.1 -1 ,000 nM were observed. These experimentally-derived binding curves aligned quite well with the two-regime binding predicted by the model. PANDAS_ds6 and PANDAS_ds7 approximated single-isotherm binding, with increased sensitivity as a result of antibody-assisted binding, yielding KD values of 97.72 pM and 1 .43 nM, respectively. In contrast, PANDAS_ds8 and PANDAS_ds9 exhibited two- stage binding with a broader dynamic range due to antibody-independent binding. The ability to rationally tune response from high-sensitivity to wide dynamic range sensing could enable the optimization of PANDAS constructs for specific biomolecular detection applications. Although PANDAS_ds6 exhibited the best sensitivity, its background was also high due to the less stable hybridization status. For the remainder of this work, PANDAS_ds7 was chosen for further characterization because of its low background signal, high signal gain, and relatively high thrombin sensitivity.

[0111] To evaluate the kinetic response of this construct, PANDAS_ds7-coupled beads were dispensed into a 1 pM thrombin-spiked buffer and followed by incubation for three minutes to allow them to equilibrate. The beads were then washed with thrombin-free buffer and the PANDAS fluorescent binding response was periodically interrogated via flow cytometry to observe the target dissociation signal (Fig. 4a). As a control, this experiment was repeated by washing the beads with the same 1 pM thrombin-containing solution, which should result in minimal dissociation. A dissociation rate of kotf = 8.3 x 104s1was measured, which indicates that the PANDAS construct maintains a reasonable off-rate that is suitable for tracking endogenous target concentration changes in continuous sensing applications, given an antibody with typical binding kinetics. Also evaluated was the reversibility of binding by deploying the PANDAS- coupled beads into alternating solutions of 1 pM thrombin and buffer solutions, incubating for 15 minutes each time, and then interrogating the construct via flow cytometry (Fig. 4b). The PANDAS receptor was able to track these changes in concentration over many cycles, with the binding signal returning to near-baseline levels every time.

[0112] Finally, the PANDAS construct’s performance as a continuous sensor was assessed in complex samples. Working with the protein-rich matrix of blood, serum, or plasma would have required a careful selection of samples to avoid the influence of proteins that interfere with the ability to accurately measure spiked-in thrombin concentrations. Endogenous antithrombin III (ATI II), heparin cofactor II (HCI I) , and alpha-2-macroglobulin (A2M) can all form complexes with thrombin and thereby interfere with the function of the PANDAS sensor26 28. A surrogate proteinrich biofluid of defined content was therefore used, challenging the switch with buffer containing physiologically representative concentrations of BSA (-530 pM) as a surrogate for human albumin — a large protein that is prone to nonspecific binding and composes 60% of the total plasma protein content29. The fluorescent response of PANDAS_ds7 was measured in this albumin-rich solution using the same bead-based 30 min fluorescent assay described above. The PANDAS response to thrombin in the presence of albumin did not differ significantly from its performance in buffer (Fig. 4c), indicating that the PANDAS design can leverage the antibody’s excellent specificity to facilitate sensitive aptamer-based detection in complex samples. This is especially notable as the TBA aptamer has been noted to have cross-reactivity to bovine serum albumin with micromolar affinity30. Finally, to test the construct’s capacity for time-resolved continuous sensing in complex samples, the beads were sequentially incubated with varying physiological concentrations of thrombin in the presence of 530 pM albumin, and the switch response was measured every 15 min (Fig. 4d). Stable concentration-dependent measurements were observed over the entire 270-minute experiment, demonstrating the potential for continuous monitoring of analyte concentrations with the PANDAS sensor.

[0113] Example 5 - Thermodynamic binding model for the PANDAS construct

[0114] It was begun with the assumption that the target concentration T > Ut, where Utis the total concentration of PANDAS in solution (initially in state U), and then assign the following Boltzmann weights in accordance with the equilibria described in Fig. 2a:

[0115] An expression for the fraction of fluorescent molecules can then be derived as a function of target, F(T), as:

[0116] Eq. S1 is plotted as the solid lines in Fig. 2. If it is assumed that KDAp, Ceff» KDAb, as can be expected from a PANDAS employing a high affinity antibody and a short linker, Eq. S1 can be approximated as the sum of two scaled isotherms, and is easily related to the qualitative interpretation provided in the main text. The first isotherm has a high affinity, with an effective dissociation constant defined by: which describes the antibody-assisted binding of the aptamer. The second isotherm has a low affinity, and is defined by KDLA= KDAp+ Ceff, which describes the un-assisted binding fraction. Note that the scaling of the isotherms depends on the relative values of KDApand Ceff. If Ceff« KDAp, then most of the binding will be un-assisted, while if Ceff» KDAp, the majority of signal will be from antibody-assisted binding. Thus, one can define the parameter a = — K , which DAp+ceff describes the fraction of antibody-assisted binding of a given PANDAS system, such that:

[0117] For the case where Ceffis large, antibody-assisted binding dominates, and a « 1. In this case, Eq. S3 approaches Eq. 1 .

[0118] Materials and Methods

[0119] Reagents and materials

[0120] DNA oligonucleotides with fluorophore-quencher modifications were purchased from Integrated DNA Technologies. Oligonucleotide concentrations were determined by UV spectroscopy using a Nanodrop ND2000 spectrophotometer. Thrombin (HCT-0020 human a- thrombin) was purchased from Haematologic Technologies. Thrombin monoclonal antibody 5020 (Catalog # MA1 -43019) was purchased from ThermoFisher. Thrombin monoclonal antibody F1 (sc-271449) was purchased from Santa Cruz Biotechnology. TNF-a antibody was purchased from Biolegend (Catalog # 502802. Protein G Dynabeads (Catalog # 10-004-D) was purchased from Invitrogen. NuPAGE sample reducing agent, NuPAGE LDS Sample Buffer (4x), NuPAGE 4 to 12% Bis-Tris mini protein gels, 20x NuPAGE MES SDS running buffer, SeeBlue pre-stained protein standard, SimplyBlue SafeStain, UltraPure TBE buffer (10X), Novex TBE-Urea Gels (10%), GelStar nucleic acid gel stain, and Tween 20 were purchased from Thermo Fisher Scientific.

[0121] Screening aptamer-antibody pairs for the PANDAS construct

[0122] Two Cy5-labeled thrombin aptamer sequences (TBA and HD22) and two different antithrombin antibodies (5020 and F1 ) were purchased. 200 nM antibody was incubated with 10 pL protein G Dynabeads in 50 pL 1 x ISD binding buffer (10 mM Tris-HCI, pH 7.5 and 6 mM MgCh) for 30 min at room temperature. The beads were washed three times to remove any nonspecific binding, and then incubated with 450 nM aptamer in the presence or absence of 300 nM thrombin in a final volume of 50 pL ISD buffer for 30 min at room temperature. It was predicted that noncompetitive pairs would form sandwich pairs that produce a fluorescence shift on beads in the presence of thrombin, which could be detected by flow cytometer. Screening results are shown in Fig. 6 and 7.

[0123] PANDAS assembly

[0124] The SiteClick antibody azido modification kit (S20026, Thermo Fisher) was used to modify the antibody Fc region in a site-specific fashion. 100 pg of antibody was treated with 10 pL 0- galactosidase in 20 pL 1 x PBS buffer at 37 °C overnight to remove the terminal 0-D-galactoside. The uncapped antibodies were then treated with 80 pL GalT transferase and UDP-GalNAz in 1 x Tris buffer at 30 °C overnight to add azide caps. 50K Amicon purification columns were used to remove excessive UDP-GalNAz. 50 pL of 100 pM amine-modified anchor oligonucleotides (Ab2- adapter) were treated with 100 pL 17 mM DBCO-NHS at room temperature overnight to equip these oligos with DBCO moieties. The oligos were then ethanol precipitated to remove excess reactants and resuspended in 1 x ISD buffer. 10 pL of 100 pM DBCO-modified anchor oligonucleotides were reacted with 10 pL of 10 pM azido-antibody in 1 x PBS buffer, and the degree of antibody labeling was determined by running a reducing NuPAGE gel (see below). The antibody-anchor complexes were stored at 4 °C until further use. To make fresh PANDAS, 2 pL of 5 pM antibody-anchor complexes was hybridized to 1 pL of 100 pM aptamers in hybridization buffer ( (1x PBS, 1 mg / mL BSA, 0.05% Tween 20, 15 pg / m goat IgG, 0.1 mg / mL salmon sperm DNA, and 5 mM EDTA)) overnight. 10 pmol of PANDAS chimera was then captured by 10 pL of prewashed protein G Dynabeads and washed three times with ISD buffer to remove excess aptamers.

[0125] Antibody labeling quantification in reducing gels

[0126] The PANDAS antibody:oligo ratio was evaluated by quantifying the degree of antibody labeling in 10% NuPAGE gels (Fig. 9). 1 pL NuPAGE reducing reagent was incubated with 12 pmol unmodified antibody or click-modified antibody in a total volume of 10 pL 1 X NuPAGE sample loading buffer at 95 °C for 5 min. The samples were then quickly loaded onto the 10% Bis-Tris mini protein gel and run at 200 V for 30 min. After 30 min, the gel was transferred to 100 mL SimplyBlue SafeStain staining solution for another 30 min. The gel was rinsed with water twice to destain before imaging on a ChemiDoc MP System from Bio-Rad Laboratories.

[0127] Biolayer interferometry (BLI) analysis of PANDAS components

[0128] BLI affinity measurements were done on an Octet Red 96 instrument (Pall ForteBio), with streptavidin sensors (SA, Sartorius) and Fc capture sensors (AMC, Sartorius). Sensors were prehydrated in ISD buffer for at least 20 min before use. 200 pL of 20 nM biotinylated aptamers or antibodies in ISD buffer were used for ligand loading to reach ~0.5 nm loading levels. After ligand loading, sensors were dipped in ISD binding buffer containing various thrombin concentrations (25, 50, 100, or 200 nM for aptamer measurements; 10, 20, 40, or 80 nM for antibody measurements) for 300 sec and then dipped in ISD buffer for 600 sec. To avoid nonspecific binding, every thrombin concentration was measured with both assay and reference sensors. Final signals were processed via signal subtraction from reference sensors.

[0129] Measurements of effective binding affinity

[0130] To obtain binding curves, 50 L reactions were prepared in 1 x ISD binding buffer with 10 nM PANDAS-coupled beads and thrombin concentrations in the range of 1 nM-1 pM for 30 min at room temperature. For ISDs alone tuning experiments, TNF-a antibody was used, an antibody which was shown have no affinity for thrombin as a dummy antibody in PANDAS. For baseline determination with the PANDAS alone, 50 pL of 10 nM PANDAS-coupled beads were prepared in ISD binding buffer without thrombin. The mean fluorescent intensities for all samples were measured at 25 °C on a flow cytometer (BD Accuri C6 Plus).

[0131] For the reversibility experiments, three samples of 10 nM PANDAS-coupled beads and 1 pM thrombin in 50 pL of 1 x ISD buffer were incubated for 30 min at room temperature to reach saturation. The sample tubes were then put in a magnetic rack (Life Technologies) for 2 min to separate PANDAS beads and sample liquid. For the first sample, saturation was maintained with 50 pL of 1 pM thrombin in ISD buffer added to the beads every 15 min. The second sample was for reversibility testing, and 50 pL of ISD buffer or 1 pM thrombin in ISD buffer were added every 15 min in an alternating fashion. The third sample was used to measure dissociation only, with 50 pL of 1 x ISD binding added to the PANDAS beads every 15 min. The mean fluorescent intensities for all samples were measured at 25 °C on a flow cytometer.

[0132] Signal normalization for thermodynamic plots

[0133] Samples were prepared in triplicate. A single-site hyperbolic binding curve was applied to fit and normalize data from the flow cytometer for ease of comparison:

[0134] The average fluorescence value was divided by Bmaxbefore plotting. Error bars were calculated as propagation of errors for standard deviation from the average fluorescence and error in the fit for Bmnr.

[0135] References

[0136] (1 ) Kumar, A.; Roberts, D.; Wood, K. E.; Light, B.; Parrillo, J. E.; Sharma, S.; Suppes, R.; Feinstein, D.; Zanotti, S.; Taiberg, L.; Gurka, D.; Kumar, A.; Cheang, M. Duration of Hypotension before Initiation of Effective Antimicrobial Therapy Is the Critical Determinant of Survival in Human Septic Shock*. Crit. Care Med. 2006, 34 (6), 1589-1596. https: / / doi.Org / 10.1097 / 01 .CCM.0000217961 .75225.E9.

[0137] (2) Keller, T.; Zeller, T.; Peetz, D.; Tzikas, S.; Roth, A.; Czyz, E.; Bickel, C.; Baldus, S.; Warnholtz, A.; Frohlich, M.; Sinning, C. R.; Eleftheriadis, M. S.; Wild, P. S.; Schnabel, R. B.; Lubos, E.; Jachmann, N.; Genth-Zotz, S.; Post, F.; Nicaud, V.; Tiret, L.; Lackner, K. J.; Munzel, T. F.; Blankenberg, S. Sensitive Troponin I Assay in Early Diagnosis of Acute Myocardial Infarction. N. Engl. J. Med. 2009, 361 (9), 868-877. https: / / doi.Org / 10.1056 / NEJMoa0903515. (3) Vettoretti, M.; Cappon, G.; Acciaroli, G.; Facchinetti, A.; Sparacino, G. Continuous Glucose Monitoring: Current Use in Diabetes Management and Possible Future Applications. J. Diabetes Sci. Technol. 2018, 12 (5), 1064-1071. https: / / doi.Org / 10.1177 / 1932296818774078.

[0138] (4) Hellhammer, D. H.; Wust, S.; Kudielka, B. M. Salivary Cortisol as a Biomarker in Stress Research. Psycho neuroendocrinology 2009, 34 (2), 163-171. https: / / d0i.0rg / l 0.1016 / j.psyneuen.2008.10.026.

[0139] (5) Bhake, R. C.; Kluckner, V.; Stassen, H.; Russell, G. M.; Leendertz, J.; Stevens, K.; Linthorst, A. C. E.; Lightman, S. L. Continuous Free Cortisol Profiles — Circadian Rhythms in Healthy Men. J. Clin. Endocrinol. Metab. 2019, 104 (12), 5935-5947. https: / / doi.Org / 10.1210 / JC.2019-00449.

[0140] (6) Gruenewald, T. L.; Seeman, T. E.; Ryff, C. D.; Karlamangla, A. S.; Singer, B. H. Combinations of Biomarkers Predictive of Later Life Mortality. Proc. Natl. Acad. Sci. U. S. A. 2006, 703 (38), 14158-14163. https: / / doi.org / 10.1073 / pnas.0606215103.

[0141] (7) Kim, J.; Campbell, A. S.; de Avila, B. E.-F.; Wang, J. Wearable Biosensors for Healthcare Monitoring. Nat. Biotechnol. 2019, 37(4), 389-406. https: / / doi.org / 10.1038 / s41587-019- 0045-y.

[0142] (8) Aydin, S. A Short History, Principles, and Types of ELISA, and Our Laboratory Experience with Peptide / Protein Analyses Using ELISA. Peptides 2015, 72, 4-15. https: / / d0i.0rg / l 0.1016 / j . peptides.2015.04.012.

[0143] (9) Wu, C.; Dougan, T. J.; Walt, D. R. High-Throughput, High-Multiplex Digital Protein Detection with Attomolar Sensitivity. ACS Nano 2022, 76 (1), 1025-1035. https: / / doi.org / 10.1021 / acsnano.1c08675.

[0144] (10) Das, J.; Gomis, S.; Chen, J. B.; Yousefi, H.; Ahmed, S.; Mahmud, A.; Zhou, W.; Sargent, E. H.; Kelley, S. O. Reagentless Biomolecular Analysis Using a Molecular Pendulum. Nat. Chem. 2021 , 73 (5), 428-434. https: / / doi.org / 10.1038 / s41557-021 -00644-y.

[0145] (11 ) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Label-Free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer-Based Sensor. Angew. Chem. Int. Ed. 2005, 44 (34), 5456-5459. https: / / doi.org / 10.1002 / anie.200500989.

[0146] (12) Swensen, J. S.; Xiao, Y.; Ferguson, B. S.; Lubin, A. A.; Lai, R. Y.; Heeger, A. J.; Plaxco, K. W.; Soh, H. Tom. Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor. J. Am. Chem. Soc. 2009, 131 (12), 4262-4266. https: / / doi.org / 10.1021 / ja806531z.

[0147] (13) Ferguson, B. S.; Hoggarth, D. A.; Maliniak, D.; Ploense, K.; White, R. J.; Woodward, N.; Hsieh, K.; Bonham, A. J.; Eisenstein, M.; Kippin, T. E.; Plaxco, K. W.; Soh, H. T. Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals. Sci. Transl. Med. 2013, 5 (213). https: / / doi.org / 10.1126 / scitranslmed.3007095.

[0148] (14) Nakatsuka, N.; Yang, K.-A.; Abendroth, J. M.; Cheung, K. M.; Xu, X.; Yang, H.; Zhao, C.; Zhu, B.; Rim, Y. S.; Yang, Y.; Weiss, P. S.; Stojanovic, M. N.; Andrews, A. M. Aptamer- Field-Effect Transistors Overcome Debye Length Limitations for Small-Molecule Sensing. Science 2018, 362 (6412), 319-324. https: / / doi.org / 10.1126 / science.aao6750.

[0149] (15) Dauphin-Ducharme, P.; Yang, K.; Arroyo-Curras, N.; Ploense, K. L.; Zhang, Y.; Gerson, J.; Kurnik, M.; Kippin, T. E.; Stojanovic, M. N.; Plaxco, K. W. Electrochemical Aptamer- Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback- Controlled Drug Delivery. ACS Sens. 2019, 4 (10), 2832-2837. https: / / doi.org / 10.1021 / ACSSENSORS.9B01616 / SUPPL_FILE / SE9B01616_SI_001.PDF.

[0150] (16) Tang, Z.; Mallikaratchy, P.; Yang, R.; Kim, Y.; Zhu, Z.; Wang, H.; Tan, W. Aptamer Switch Probe Based on Intramolecular Displacement. J. Am. Chem. Soc. 2008, 130 (34), 11268-11269. https: / / doi.Org / 10.1021 / ja804119s.

[0151] (17) Wilson, B. D.; Hariri, A. A.; Thompson, I. A. P.; Eisenstein, M.; Soh, H. T. Independent Control of the Thermodynamic and Kinetic Properties of Aptamer Switches. Nat. Commun. 2019, 70 (1 ), 5079. https: / / doi.org / 10.1038 / s41467-019-13137-x.

[0152] (18) Munzar, J. D.; Ng, A.; Juncker, D. Duplexed Aptamers: History, Design, Theory, and Application to Biosensing. Chem. Soc. Rev. 2019. https: / / doi.org / 10.1039 / C8CS00880A.

[0153] (19) Liu, T.; Qian, W.-J.; Gritsenko, M. A.; Xiao, W.; Moldawer, L. L.; Kaushal, A.; Monroe, M. E.; Varnum, S. M.; Moore, R. J.; Purvine, S. O.; Maier, R. V.; Davis, R. W.; Tompkins, R. G.; Camp, D. G.; Smith, R. D. High Dynamic Range Characterization of the Trauma Patient Plasma Proteome*. Mol. Cell. Proteomics 2006, 5 (10), 1899-1913. https: / / doi.Org / 10.1074 / mcp.M600068-MCP200.

[0154] (20) Tasset, D. M.; Kubik, M. F.; Steiner, W. Oligonucleotide Inhibitors of Human Thrombin That Bind Distinct Epitopes. J. Mol. Biol. 1997, 272 (5), 688-698. https: / / d0i.0rg / l 0.1006 / jmbi.1997.1275.

[0155] (21) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. J. Selection of Single- Stranded DNA Molecules That Bind and Inhibit Human Thrombin. Nature 1992, 355 (6360), 564-566. https: / / doi.Org / 10.1038 / 355564a0.

[0156] (22) Kong, D.; Lei, Y.; Yeung, W.; Hili, R. Enzymatic Synthesis of Sequence-Defined Synthetic Nucleic Acid Polymers with Diverse Functional Groups. Angew. Chem. 2016, 728 (42), 13358-13362.

[0157] (23) Kong, D.; Yeung, W.; Hili, R. In Vitro Selection of Diversely Functionalized Aptamers. J. Am. Chem. Soc. 2017, 739 (40), 13977-13980.

[0158] (24) Prager, N. A.; Abendschein, D. R.; McKenzie, C. R.; Eisenberg, P. R. Role of Thrombin Compared with Factor Xa in the Procoagulant Activity of Whole Blood Clots. Circulation 1995, 92 (4), 962-967. https: / / doi.Org / 10.1161 / 01.cir.92.4.962.

[0159] (25) Estevez, B.; Kim, K.; Delaney, M. K.; Stojanovic-Terpo, A.; Shen, B.; Ruan, C.; Cho, J.; Ruggeri, Z. M.; Du, X. Signaling-Mediated Cooperativity between Glycoprotein Ib-IX and Protease-Activated Receptors in Thrombin-Induced Platelet Activation. Blood 2016, 727(5), 626-636. https: / / doi.Org / 10.1 182 / blood-2015-04-638387.

[0160] (26) Olson, S. T.; Chuang, Y.-J. Heparin Activates Antithrombin Anticoagulant Function by Generating New Interaction Sites (Exosites) for Blood Clotting Proteinases. Trends Cardiovasc. Med. 2002, 12 (8), 331-338. https: / / doi.org / 10.1016 / S1050-1738(02)00183-4.

[0161] (27) Tollefsen, D. M. Heparin Cofactor II Modulates the Response to Vascular Injury. Arterioscler. Thromb. Vase. Biol. 2007, 27 (3), 454-460. https: / / doi.org / 10.1 161 / 01.ATV.0000256471 .22437.88.

[0162] (28) Straight, D. L.; McKee, P. A. Characterization of Thrombin Binding to Alpha 2- Macroglobulin. J. Biol. Chem. 1984, 259 (2), 1272-1278. https: / / doi.org / 10.1016 / S0021- 9258(17)43599-X.

[0163] (29) He, X. M. ; Carter, D. C. Atomic Structure and Chemistry of Human Serum Albumin. Mature 1992, 358 (6383), 209-215. https: / / doi.org / 10.1038 / 358209a0.

[0164] (30) Trapaidze, A.; Herault, J.-P.; Herbert, J.-M.; Bancaud, A.; Gue, A.-M. Investigation of the Selectivity of Thrombin-Binding Aptamers for Thrombin Titration in Murine Plasma. Biosens. Bioelectron. 2016, 78, 58-66. https: / / doi.Org / 10.1016 / j.bios.2015.11 .017.

[0165] (31 ) Hariri, A. A.; Cartwright, A. P.; Dory, C.; Gidi, Y.; Yee, S.; Fu, K. X.; Yang, K.; Wu, D. ; Thompson, I. A. P.; Maganzini, N.; Feagin, T.; Young, B. E.; Afshar, B. H.; Eisenstein, M.; Digonnet, M.; Vuckovic, J.; Soh, H. T. Continuous Optical Detection of Small-Molecule Analytes in Complex Biomatrices. bioRxiv March 6, 2023, p 2023.03.03.531030. https: / / doi.org / 10.1 101 / 2023.03.03.531030.

[0166] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1. A molecular sensor comprising: a capture agent that binds to a first epitope of an analyte; and an aptamer that binds to a second epitope of the analyte, wherein the aptamer is stably associated with the capture agent, and wherein the aptamer produces a detectable signal upon cooperative binding of the capture agent and aptamer to the analyte.

2. The molecular sensor of claim 1 , wherein the capture agent is an antibody that binds the analyte.

3. The molecular sensor of claim 2, wherein the antibody is an IgG, Fab, scFv, scFv-Fc, scFv-CH3, scFv-zipper, scFab, VHH / VH, or diabody.

4. The molecular sensor of claim 1 , wherein the capture agent is a T-cell receptor (TCR) that binds the analyte.

5. The molecular sensor of claim 1 , wherein the analyte is a receptor and the capture agent is a ligand of the receptor.

6. The molecular sensor of claim 1 , wherein the analyte is a ligand and the capture agent is a receptor for the ligand.

7. The molecular sensor of any one of claims 1 to 6, wherein the aptamer is linked to a displacement strand (DS) domain complementary to a binding motif of the aptamer that binds to the analyte, and wherein the DS domain is displaced upon cooperative binding of the capture agent and aptamer to the analyte.

8. The molecular sensor of any one of claims 1 to 6, wherein the aptamer is hybridized to a domain of a displacement strand (DS) complementary to a binding motif of the aptamer that binds to the analyte, wherein the domain of the DS is displaced upon cooperative binding of the capture agent and aptamer to the analyte, and wherein the aptamer and DS are separate nucleic acid molecules.

9. The molecular sensor of claim 7 or 8, wherein the detectable signal is produced upon displacement of the DS domain.

10. The molecular sensor of any one of claims 1 to 9, wherein the detectable signal is a fluorescent signal.11 . The molecular sensor of claim 10, wherein the aptamer is labeled with a fluorophore.

12. The molecular sensor of claim 11 , wherein the aptamer comprises a quencher moiety that quenches the fluorophore in a first conformation when the molecular sensor is not bound to the analyte, and wherein upon cooperative binding of the capture agent and aptamer to the analyte, the aptamer assumes a second confirmation in which the fluorophore assumes an unquenched state.

13. The molecular sensor of any one of claims 1 to 9, wherein the detectable signal is an electrochemical signal.

14. The molecular sensor of any one of claims 1 to 13, wherein the aptamer is stably associated with the capture agent through a covalent linkage.

15. The molecular sensor of claim 14, wherein the molecular sensor is a capture agentaptamer conjugate.

16. The molecular sensor of claim 15, wherein the capture agent comprises a polypeptide site-specif ically conjugated to the aptamer.

17. The molecular sensor of claim 16, wherein the capture agent is an antibody comprising a constant region, and wherein the aptamer is site-specifically conjugated to the constant region.

18. The molecular sensor of claim 17, wherein the constant region is an Fc region.

19. The molecular sensor of any one of claims 1 to 13, wherein the aptamer is stably associated with the capture agent through a non-covalent linkage.

20. The molecular sensor of claim 19, wherein the capture agent comprises a nucleic acid anchor, wherein the aptamer comprises a domain complementary to the nucleic acid anchor, and wherein the aptamer is stably associated with the capture agent via hybridization between the nucleic acid anchor and the domain complementary to the nucleic acid anchor.21 . The molecular sensor of claim 20, wherein the capture agent comprises a polypeptide site-specifically modified with the nucleic acid anchor.

22. The molecular sensor of claim 21 , wherein the capture agent is an antibody comprising a constant region, and wherein the constant region is site-specifically modified with the nucleic acid anchor.

23. The molecular sensor of claim 22, wherein the constant region is an Fc region.

24. A substrate comprising the molecular sensor of any one of claims 1 to 23 immobilized on the surface thereof.

25. The substrate of claim 24, comprising a plurality of the molecular sensors.

26. The substrate of claim 24 or 25, wherein the substrate is comprised within a well or channel.

27. The substrate of claim 24 or 25, wherein the substrate is a microparticle.

28. The substrate of claim 27, wherein the microparticle is a bead.

29. The substrate of claim 24 or 25, wherein the substrate is a hydrogel, a microneedle or an optical fiber.

30. A device comprising the molecular sensor of any one of claims 1 to 23.31 . The device of claim 30, comprising the substrate of any one of claims 24 to 29.

32. The device of claim 30 or 31 , wherein the device is a microfluidic device.

33. The device of claim 30 or 31 , wherein the device is a lateral flow device.

34. The device of claim 30 or 31 , wherein the device is a test strip.

35. The device of claim 30 or 31 , wherein the device is a microneedle patch.

36. A method of assessing a sample for an analyte, the method comprising: contacting the molecular sensor of any one of claims 1 to 23 with the sample under conditions suitable for cooperative binding of the capture agent and aptamer to the analyte; and assessing for a detectable signal produced by the aptamer.

37. The method according to claim 36, wherein the contacting comprises contacting the substrate of any one of claims 24 to 29 with the sample.

38. The method according to claim 36 or 37, wherein the sample is a biological sample obtained from a subject.

39. The method according to claim 38, wherein the biological sample is a biological fluid.

40. The method according to claim 39, wherein the biological fluid is blood or a fraction thereof, urine, saliva, interstitial fluid (ISF) , cerebrospinal fluid, tears, mucus, or sputum.41 . The method according to any one of claims 36 to 40, wherein the analyte is a biomarker.

42. The method according to claim 41 , wherein the biomarker is a disease biomarker.

43. The method according to any one of claims 36 to 40, wherein the analyte is a therapeutic agent or metabolite thereof.

44. The method according to any one of claims 36 to 40, wherein the analyte is a pathogen.

45. The method according to claim 44, wherein the pathogen is a virus, a bacterium, a fungus, a protozoan, or a worm.

46. The method according to any one of claims 36 to 40, wherein the analyte is a drug of abuse or metabolite thereof.

47. The method according to any one of claims 36 to 46, comprising assessing for the analyte at two or more time points.

48. The method according to claim 47, wherein the same sample is assessed at the two or more time points.

49. The method according to claim 47, comprising assessing iterative samples for the analyte.

50. The method according to any one of claims 36 to 49, wherein the method is implemented using the device of any one of claims 30 to 35.

51. A kit comprising: the molecular sensor of any one of claims 1 to 23; and instructions for using the molecular sensor to assess a sample for an analyte.

52. The kit of claim 51 , comprising the substrate of any one of claims 24 to 29.

53. The kit of claim 51 or claim 52, comprising the device of any one of claims 30 to 35.

54. The kit of any one of claims 51 to 53, wherein the instructions comprise instructions for performing the method according to any one of claims 36 to 49.

Images