Systems and methods for analyte detection using toehold-mediated strand displacement

Abstract

The present disclosure provides systems, kits, and platforms for detecting analytes in samples using toehold-mediated strand displacement. Methods of using the systems, kits, and platforms are also provided.

Description

SYSTEMS AND METHODS FOR ANALYTE DETECTION USING TOEHOLD- MEDIATED STRAND DISPLACEMENTCROSS-REFERENCE TO RELATED APPLCIATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63 / 440,513, filed January 23, 2023, the content of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (121892.00134.xml; Size: 21,907 bytes; and Date of Creation: December 22, 2023) is herein incorporated by reference in its entirety.BACKGROUND

[0003] The invention described herein relates to systems and methods for detecting analytes in a biological sample. Some common detection methods utilize a reagent or kit of reagents including a first reagent comprising an analyte binding protein conjugated to single-stranded DNA (ssDNA); and a second reagent comprising a fluorochrome coupled to a ssDNA that is complementary to the ssDNA of the first reagent. Observing fluorescence of the fluorochrome allows for detection of a target analyte bound to the analyte binding protein. The hybridization of complementary ssDNA is highly specific. However, nonspecific binding of the ssDNA to the sample can occur. Consequently, false positive fluorescent signals make high-quality data acquisition very difficult. The non-specific binding of fluorophore-ssDNA can be reduced by complicated blocking steps or washing with stringent buffers, which can have a negative influence on the sample integrity and epitope detection. Therefore, reagents and methods for detecting analytes in a sample with improved sensitivity and specificity are needed.SUMMARY

[0004] Systems, kits, and platforms for detecting analytes in a sample are provided herein. In one embodiment, the system, kit, or platform comprises at least one analyte binding molecule, the analyte binding molecule comprising an analyte binding moiety, and a nucleic acid invader molecule, the nucleic acid invader molecule comprising a toehold region and a main region,wherein the analyte binding moiety is linked to the invader molecule; a nucleic acid detection reagent comprising a dye strand, the dye strand comprising a dye sequence that is complementary to the toehold region and the main region of the invader molecule, and a dye molecule of a dyeacceptor pair; and an acceptor strand, the acceptor strand comprising an acceptor sequence that comprises the main region, and an acceptor molecule of the dye-acceptor pair; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule.

[0005] In another embodiment, the system, kit, or platform comprises at least one analyte binding molecule comprising an analyte binding moiety linked to a nucleic acid invader molecule comprising a target sequence; a nucleic acid detection reagent comprising a dye sequence on a first end, a dye molecule of a dye-acceptor pair linked to the dye sequence, an acceptor sequence on a second end, an acceptor molecule of the dye-acceptor pair linked to the acceptor sequence, and a detection sequence between the dye sequence and the acceptor sequence, wherein the dye sequence and the acceptor sequence are complementary to each other; wherein the detection sequence is complementary to the target sequence; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; and wherein when the detection sequence hybridizes to the target sequence, the acceptor molecule is unable to suppress the signal emitted by the dye molecule.

[0006] In another embodiment, the system, kit, or platform, comprises at least one analyte binding molecule comprising an analyte binding moiety, and a nucleic acid analyte strand, the nucleic acid analyte strand comprising a dye target region; and an acceptor target region, wherein the analyte binding moiety is linked to the analyte strand; a nucleic acid dye strand comprising a dye sequence that is complementary to the dye target region, and a dye molecule of a dye-acceptor pair; a nucleic acid acceptor strand comprising an acceptor sequence that is complementary to the acceptor target region, a toehold sequence, and an acceptor molecule of the dye-acceptor pair; and a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; wherein when the dye sequence and the acceptor sequence hybridize to the analyte strand, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; and wherein when the invader molecule hybridizes to the acceptor strand, the acceptor molecule is unable to suppress the signal.

[0007] In another embodiment, the system, kit, or platform comprises at least one analyte binding molecule comprising an analyte binding moiety, and a nucleic acid analyte strand, wherein the analyte binding moiety is linked to the analyte strand; a nucleic acid dye strand comprising, an analyte target region that is complementary to the analyte strand, a dye sequence, and a dye molecule of a dye-acceptor pair; and a nucleic acid acceptor strand comprising an acceptor sequence that is complementary to the dye sequence, a toehold sequence, and an acceptor molecule of the dye-acceptor pair; and a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; wherein when the analyte target region hybridizes to the analyte strand, a signal is emitted by the dye molecule; wherein when the acceptor strand hybridizes to the dye sequence, the acceptor molecule is positioned to suppress the signal; and wherein when the invader molecule hybridizes to the acceptor strand, the signal is emitted.

[0008] The foregoing and other aspects and advantages of the invention will appear from the following description. In the description, reference is made to the accompanying drawings which form a part hereof, and in which there is shown by way of illustration preferred embodiments of the invention. Such embodiments do not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and description herein for interpreting the scope of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] FIG. 1 is a schematic illustrating the principle of toehold-mediated strand displacement (TMSD).

[0011] FIG. 2 illustrates exemplary reagents used in the systems and methods described herein.

[0012] FIG. 3A-E illustrate the switching on and off of an analyte binding molecule in a first configuration of the systems and methods described herein.

[0013] FIG. 4 illustrates a first configuration of the systems described herein.

[0014] FIG. 5 illustrates a second configuration of the systems described herein.

[0015] FIG. 6 illustrates a third configuration of the systems described herein.

[0016] FIG. 7 illustrates a modification of the third configuration of the systems described herein.

[0017] FIG. 8 illustrates a fourth configuration of the systems described herein.

[0018] FIG. 9 illustrates a fifth configuration of the systems describe herein.

[0019] FIG. 10A-D. shows fluorescence profiles using the system of FIGs. 3. A) Fluorescence signal (y-axis) of the dye-strand is measured over time (x-axis). B) A 2x molar excess of the acceptor strand was added and after 30 sec and the fluorescence signal was measured as before. After around 2:30 min the signal reaches a minimum and the dye signal is nearly completely suppressed. C) The invader strand is added in an equimolar amount as the dye strand. After 30 sec the fluorescence signal was measured as before. After around 3:30 min the signal reaches a maximum. The acceptor strand is displaced by the invader strand and the fluorescence signal of the dye can be measured again. D) The acceptor fragment is added to the sample in a 2x molar excess to the dye strand. After 30 sec the fluorescence signal is measured and after 3:00 min the signal reaches a minimum, because the acceptor suppresses the dye signal. The results represent the measurement of three independent experiments. The experiment was further visualized by pictures of the samples after every step under black light which excites the fluorophore. The experiment represents the system shown in Fig.3 but without the antibody.

[0020] FIG. 11A-B: Non-specific staining of not suppressed detection probes. A) shows fluorescence picture of human PBMCs before incubation with a not suppressed detection probe. B) shows fluorescence picture of the same PBMCs as in 1 after 5 min incubation with a not suppressed detection probe.

[0021] Fig. 12A-D: Experimental data for a system as shown in Figure 3. Fluorescence microscopy pictures of human PBMCs incubated with an antibody targeting CD4 (clone RPA-T4) and CD45 (clone HI30). Both antibodies were conjugated to an invader strand with distinct toe sequence. Cells incubated with both antibodies, panel (A) The detection probe for CD4 was addedto the cells and incubated for 5 min. CD4 positive cells can be detected by the fluorescence signal in channel 1, panel (B and C). The detection probe for CD45 was added to the cells and incubated for 5 min. CD45 positive cells can be detected by the fluorescence signal in channel 2 panel (C). Due to the signal suppression of the detection probe, the unspecific binding of the DNA leads to no unspecific signal, panel (C) The acceptor fragment is added to the cells and incubated for 5 min. The fluorescence signal of both antibodies is suppressed, panel (D).

[0022] FIG. 13A-D: Experimental data for a system as shown in Figure 9. Fluorescence microscopy pictures of human PBMCs incubated with an antibody targeting CD4 (clone RPA-T4) and CD45 (clone HI30). Both antibodies were conjugated to an invader strand with distinct toe sequences. Cells incubated with both antibodies, panel (A) The detection probe for CD4 was added to the cells and incubated for 5 min. CD4 positive cells can be detected by the fluorescence signal in channel 1, panel (B and C). The detection probe for CD45 was added to the cells and incubated for 5 min. CD45 positive cells can be detected by the fluorescence signal in channel 2, panel (C). Due to the signal suppression of the detection probe, the unspecific binding of the DNA leads to no unspecific signal, panel (C). The acceptor fragment is added to the cells and incubated for 5 min. The fluorescence signal of both antibodies is suppressed, panel (D).DETAILED DESCRIPTION

[0023] Disclosed herein are novel systems, kits, platforms, and methods for detecting analytes in fixed cell and tissue samples. The systems and methods described herein employ toehold- mediated strand displacement (TSMD) strategies to improve specificity and sensitivity of in situ analyses of multiple analytes.

[0024] The general principle of TMSD is outlined in FIG. 1. In TMSD, a partially double stranded DNA includes a “main strand” having a relatively short single stranded “toe” region, and a complementary strand. A single stranded “invader strand” having complementarity to the main strand and the toe region, is able to hybridize to the single-stranded toe region and displace the complimentary strand and fully hybridize to the main strand to form a fully double stranded DNA.

[0025] The systems and methods disclosed herein use placement of fluorescent and acceptor molecules (or FRET pairs) on particular TMSD nucleic acid molecules to provide ON / OFFfunctionality while assessing analytes. FIG. 2 illustrates an exemplary placement of fluorophores and acceptors on nucleic acid reagents for detecting analytes. In FIG. 2, the invader strand is linked to an analyte binding moiety (AB). The invader strand includes a main region and a toe region. The dye strand is complementary to the invader strand, including the toe region, and includes a dye molecule. The dye strand also includes an overhang sequence adjacent to the sequence complementary to the invader strand main region. The first acceptor strand (acceptor strand 1) is partially complementary to the dye strand, and includes an acceptor molecule, such that when the dye strand and first acceptor strand hybridize, the signal emitted by the dye molecule is suppressed. The second acceptor strand (acceptor strand 2) is complementary to the overhang region of the dye strand, and includes the acceptor molecule, such that when the second acceptor strand and the dye strand hybridize, the signal emitted by the dye molecule is suppressed. These reagents, and variations thereof, are employed in various different configurations. Exemplary configurations of the systems and methods are described in turn below. While the configurations are named "first," "second," third," etc., this naming convention is only for convenience.

[0026] First Configuration

[0027] As illustrated in FIGs. 3A-E, in an embodiment, provided herein is a first configuration of a system, kit, or platform comprising: at least one analyte binding molecule comprising an analyte binding moiety, and a nucleic acid invader molecule, the nucleic acid invader molecule comprising a toehold region and a main region, wherein the analyte binding moiety is linked to the invader molecule; a nucleic acid detection reagent comprising a dye strand, the dye strand comprising a dye sequence that is complementary to the toehold region and the main region of the invader molecule, and a dye molecule of a dye-acceptor pair; and an acceptor strand, the acceptor strand comprising an acceptor sequence that comprises the main region, and an acceptor molecule of the dye-acceptor pair; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule.

[0028] In some embodiments, the invader molecule is single-stranded. In some embodiments, the invader molecule has double-stranded portions, and only the toehold region is single-stranded, and main region are single-stranded. In some embodiments, the invader molecule has doublestranded portions, and at least the toehold region and main region are single-stranded.

[0029] In some embodiments, the dye strand and the acceptor strand of the detection reagent are separate nucleic acid strands. In some embodiments, the dye strand and the acceptor strand are on the same nucleic acid strand, which forms a secondary structure when the dye strand and acceptor strand are hybridized to each other.

[0030] In some embodiments, the dye sequence further comprises a single-stranded overhang sequence adjacent to the dye molecule; and the system, kit, or platform further comprises an acceptor fragment that comprises the acceptor molecule and a sequence that is complementary to the overhang sequence, wherein when the acceptor fragment hybridizes to the overhang sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule. In other embodiments, the invader molecule further comprises a single-stranded overhang sequence that is not complementary to the dye strand; and the system, kit, or platform further comprises an acceptor fragment that comprises the acceptor molecule and a sequence that is complementary to the overhang sequence, wherein when the acceptor fragment hybridizes to the overhang sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

[0031] Turning again to FIG. 3, the state of the system in “OFF” mode is illustrated in FIG. 3A. The analyte binding molecule includes an analyte binding molecule (antibody) linked to a nucleic acid invader molecule (invader strand). The detection reagent includes a dye strand having a sequence complimentary to the main region and the toehold region, as well as an overhang sequence at the opposite end from the toe sequence; and an acceptor strand (acceptor strand 1). The dye strand includes a dye molecule (dye), and the acceptor strand includes an acceptor molecule, which are positioned in close proximity such that a signal emitted by the dye is suppressed, and therefore, the system is “OFF”.

[0032] FIG. 3B illustrates the binding of the toehold sequence to the dye strand, and the initiation of TMSD. In FIG. 3C, the TMSD is completed, and the acceptor strand is separated from the dye strand. Therefore, the acceptor molecule is no longer in position to suppress the dye signal, the signal is emitted, and the system is “ON.” In FIG. 3D, an acceptor fragment is introduced to the system. The acceptor fragment comprises the acceptor molecule and a sequence that is complementary to the overhang sequence of the dye strand. This initiates a switching off of the system. In FIG. 3E, the acceptor fragment has hybridized to the overhang sequence, and the acceptor molecule is able to suppress the dye signal, therefore the system is “OFF.”

[0033] FIG. 4 illustrates a modification of the first configuration of the system in which the invader strand has an overhang sequence. When the system is “ON,” an acceptor fragment can hybridize to the overhang sequence and suppress the dye signal.

[0034] In some embodiments, the system, kit, or platform comprises a plurality of analyte binding molecules. In some embodiments, each analyte binding molecule has a different binding moiety, which binds to a different analyte. The toe hold regions and main regions may be the same or different for each analyte binding molecule. In some embodiments, each analyte binding molecule has a different toe hold region on the invader molecule. In some embodiments in which each analyte binding molecule has a different toe hold region, the main regions are also different. In other embodiments in which each analyte binding molecule has a different toe hold region, the main regions are the same. In some embodiments, each nucleic acid detection reagent comprises a different dye-acceptor pair.

[0035] In some embodiments, the system is assembled into a kit. In embodiments, the kit comprises, as separated components, (a) the analyte binding molecule, (b) the detection reagent, and (c) the acceptor fragment.

[0036] In an embodiment, provided herein is a method of using the system, kit, or platform for detecting an analyte in a sample. In some embodiments, the method comprises: adding to the sample the analyte binding molecule and the nucleic acid detection reagent of the first configuration;incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

[0037] In some embodiments, the method further comprises adding to the sample the acceptor fragment; and detecting the signal.

[0038] Second Configuration

[0039] FIG. 5 illustrates a second configuration of a system, kit, or platform, which comprises: at least one analyte binding molecule comprising an analyte binding moiety linked to a nucleic acid invader molecule comprising a target sequence; a nucleic acid detection reagent comprising a dye sequence on a first end, a dye molecule of a dyeacceptor pair linked to the dye sequence, an acceptor sequence on a second end, an acceptor molecule of the dye-acceptor pair linked to the acceptor sequence, and a detection sequence between the dye sequence and the acceptor sequence, wherein the dye sequence and the acceptor sequence are complementary to each other; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; and wherein when the detection sequence hybridizes to the target sequence, the acceptor molecule is unable to suppress the signal emitted by the dye molecule.

[0040] As illustrated in FIG. 5, when the dye sequence and the acceptor sequence hybridize to each other, the detection reagent assumes a molecular beacon structure having a loop and a stem. This represents an “OFF” status of the system. The loop can serve as a toe hold sequence for initiating binding to the invader molecule. When the detection sequence binds to the target sequence, the system switches to “ON” status. In preferred embodiments, the stem portion of the molecular beacon structure is longer than 7 nucleotides to prevent dissociation of the dye sequence from the acceptor sequence under higher temperatures, e.g. >30°C.

[0041] In an embodiment, the system further comprises an acceptor fragment comprising the acceptor molecule and a sequence that this complementary to the dye sequence; wherein when the acceptor fragment hybridizes to the dye sequence, the acceptor molecule suppresses the signal emitted by the dye molecule. Thereby, the system switches to “OFF” status.

[0042] In some embodiments, the system, kit, or platform comprises a plurality of analyte binding molecules. In some embodiments, each analyte binding molecule has a different binding moiety, which binds to a different analyte. The target sequence may be the same or different for each analyte binding molecule. In some embodiments, each detection reagent comprises a different dye-acceptor pair.

[0043] In some embodiments, the system is assembled into a kit. In embodiments, the kit comprises, in separated components, (a) the analyte binding molecule, (b) the detection reagent, and (c) the acceptor fragment.

[0044] In an embodiment, provided herein is a method of using the system, kit, or platform for detecting an analyte in a sample. In some embodiments, the method comprises adding to the sample the analyte binding molecule and the nucleic acid detection reagent of the second configuration; incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

[0045] In some embodiments, the method further comprises adding to the sample the acceptor fragment; and detecting the signal.

[0046] Third Configuration

[0047] FIG. 6 illustrates a third configuration of the system, kit, or platform, which comprises:at least one analyte binding molecule comprising an analyte binding moiety, and a nucleic acid analyte strand, the nucleic acid analyte strand comprising a dye target region; and an acceptor target region, wherein the analyte binding moiety is linked to the analyte strand; a nucleic acid dye strand comprising a dye sequence that is complementary to the dye target region, and a dye molecule of a dye-acceptor pair; a nucleic acid acceptor strand comprising an acceptor sequence that is complementary to the acceptor target region, a toehold sequence, and an acceptor molecule of the dye-acceptor pair; and a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; wherein when the dye sequence and the acceptor sequence hybridize to the analyte strand, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; and wherein when the invader molecule hybridizes to the acceptor strand, the acceptor molecule is unable to suppress the signal.

[0048] As illustrated in FIG. 6, when the dye strand and the acceptor strand hybridize to the analyte strand, the acceptor molecule is able to suppress the signal emitted by the dye. This represents an “OFF” status of the system. When the invader molecule binds to the toe hold sequence of the acceptor strand, TMSD separates the acceptor strand from the analyte strand and the system is switched “ON.”

[0049] In an embodiment, the system further comprises an acceptor fragment comprising the acceptor molecule and a sequence that this complementary to the acceptor target region; wherein when the acceptor fragment hybridizes to the acceptor target region, the acceptor molecule suppresses the signal emitted by the dye molecule. Thereby, the system switches to “OFF” status.

[0050] FIG. 7 illustrates a modification of the third configuration of the system in which the dye strand has a second overhang sequence. When the system is “ON,” a second invader molecule having an acceptor molecule and a second invader sequence that is complementary to the dye sequence fragment binds to the second toe hold sequence, separates the dye strand from the analytestrand by TMSD. The proximity of the acceptor molecule to the dye molecule switches the system “OFF.”

[0051] In some embodiments, the system, kit, or platform comprises a plurality of analyte binding molecules. In some embodiments, each analyte binding molecule has a different binding moiety, which binds to a different analyte. The toe hold sequences and acceptor sequences may be the same or different for each acceptor strand. In some embodiments, each acceptor strand has a different toe hold sequence. In some embodiments in which each acceptor strand has a different toe hold sequence, the acceptor sequences are also different. In other embodiments in which each acceptor strand has a different toe hold sequence, the acceptor sequences are the same. Similarly, in embodiments comprising a dye strand having a second toe hold sequence, the toe hold sequences and dye sequences may be the same or different for each dye strand. In some embodiments, each dye strand has a different second toe hold sequence. In some embodiments in which each dye strand has a different second toe hold sequence, the dye sequences are also different. In other embodiments in which each dye strand has a different second toe hold sequence, the dye sequences are the same. In some embodiments, each dye strand and acceptor strand pair that hybridize to analyte strand comprise a different dye-acceptor pair.

[0052] In some embodiments, the system is assembled into a kit. In embodiments, the kit comprises, in separated components, (a) the analyte binding molecule, the dye strand, and the acceptor strand, (b) the invader molecule, and (c) the acceptor fragment and / or (d) the second invader molecule.

[0053] In an embodiment, provided herein is a method of using the system, kit, or platform for detecting an analyte in a sample. In some embodiments, the method comprises adding to the sample the analyte binding molecule, the dye strand, and the acceptor strand of the third configuration; incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

[0054] In some embodiments, the method further comprises adding to the sample the invader molecule; and detecting the signal. In some embodiments, the method further comprises adding to the sample the second invader molecule; and detecting the signal.

[0055] Fourth Configuration

[0056] FIG. 8 illustrates a fourth configuration of the system, kit, or platform, which comprises at least one analyte binding molecule comprising an analyte binding moiety, and a nucleic acid analyte strand, wherein the analyte binding moiety is linked to the analyte strand; a nucleic acid dye strand comprising, an analyte target region that is complementary to the analyte strand, a dye sequence, and a dye molecule of a dye-acceptor pair; and a nucleic acid acceptor strand comprising an acceptor sequence that is complementary to the dye sequence, a toehold sequence, and an acceptor molecule of the dye-acceptor pair; and a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; wherein when the analyte target region hybridizes to the analyte strand, a signal is emitted by the dye molecule; wherein when the acceptor strand hybridizes to the dye sequence, the acceptor molecule is positioned to suppress the signal; and wherein when the invader molecule hybridizes to the acceptor strand, the signal is emitted.

[0057] As illustrated in FIG. 8, when the dye strand hybridizes to the analyte strand and the acceptor strand, the acceptor molecule is able to suppress the signal emitted by the dye. This represents an “OFF” status of the system. When the invader molecule binds to the toe hold sequence of the acceptor strand, TMSD separates the acceptor strand from the analyte strand and the system is switched “ON.” In this configuration, removal of the acceptor stand from the dye strand leaves an overhang sequence on the dye strand.

[0058] In an embodiment, the system further comprises an acceptor fragment comprising the acceptor molecule and the acceptor sequence, which is complementary to the dye sequence; wherein when the acceptor fragment hybridizes to the dye sequence, the acceptor molecule suppresses the signal emitted by the dye molecule. Thereby, the system switches to “OFF” status.

[0059] In some embodiments, the system, kit, or platform comprises a plurality of analyte binding molecules. In some embodiments, each analyte binding molecule has a different binding moiety, which binds to a different analyte. The toe hold sequences and acceptor sequences may be the same or different for each acceptor strand. In some embodiments, each acceptor strand has a different toe hold sequence. In some embodiments in which each acceptor strand has a different toe hold sequence, the acceptor sequences are also different. In other embodiments in which each acceptor strand has a different toe hold sequence, the acceptor sequences are the same. In some embodiments, each dye strand and acceptor strand that hybridize to each other comprise a different dye-acceptor pair.

[0060] In some embodiments, the system is assembled into a kit. In embodiments, the kit comprises, in separated components, (a) the analyte binding molecule, the dye strand, and the acceptor strand, (b) the invader molecule, and (c) the acceptor fragment.

[0061] In an embodiment, provided herein is a method of using the system, kit, or platform for detecting an analyte in a sample. In some embodiments, the method comprises adding to the sample the analyte binding molecule, the dye strand, and the acceptor strand of the fourth configuration; incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

[0062] In some embodiments, the method further comprises adding to the sample the invader molecule; and detecting the signal. In some embodiments, the method further comprises adding to the sample the acceptor fragment; and detecting the signal.

[0063] In some embodiments, the systems and methods are used quantitatively, wherein the intensity of signal emitted from the dye correlates with the amount of analyte in the sample.

[0064] Fifth Configuration

[0065] FIG. 9 illustrates a fifth configuration of the system, kit, or platform, which comprises at least one analyte binding molecule comprising an analyte binding moiety linked to a nucleic acid invader strand comprising a toe region; a nucleic acid detection probe comprising: (a) a region complementary to the toe region of the analyte binding molecule; (b) a detection probe acceptor molecule (“D-acceptor molecule”); (c) a detection probe acceptor sequence (“D-acceptor sequence”) complementary to a first region of the invader strand of the binding molecule; (d) a loop sequence, wherein the loop sequence is complementary to a second region of the invader strand of the binding molecule; (e) a dye sequence; and (f) a dye molecule linked to the dye sequence; wherein the D-acceptor sequence is complementary to the dye sequence; wherein when the dye sequence and D-acceptor sequence hybridize to each other, the detection probe forms a molecule beacons stem-loop (e.g., hairpin) structure, and the D-acceptor molecule is positioned to suppress a signal emitted by the dye molecule; an acceptor fragment comprising an acceptor sequence and an acceptor molecule, wherein the acceptor sequence is complementary to the dye sequence of the detection probe; wherein, when regions (a), (c), and (d) of the detection probe hybridize to the invader strand of the analyte binding molecule, the D-acceptor molecule is unable to suppress the signal emitted by the dye molecule; wherein when the acceptor sequence hybridizes to the dye sequence of the detection probe, the acceptor molecule suppresses the signal emitted by the dye molecule.

[0066] As illustrated in FIG. 9, when the detection probe is not hybridized to the invader strand, the detection probe assumes a molecular beacons structure having a stem and a loop, and the D-acceptor molecule is positioned to suppress the signal emitted by the dye molecule. This represents an “OFF” status of the system. TMSD initiates the opening of the stem-loop structure of the detection probe and the preferential hybridization of regions (a), (c), and (d) of the detection probe to the invader strand. When the detection probe hybridizes to the invader strand of the analyte binding molecule, the acceptor molecule is unable to suppress the signal emitted by the dye molecule. This represents an “ON” status of the system.

[0067] In an embodiment, the system comprises an acceptor fragment comprising an acceptor molecule and an acceptor sequence that is complementary to the dye sequence. When the acceptor molecule hybridizes to the dye sequence of the detection probe, the acceptor molecule suppresses the signal emitted by the dye molecule. This also represents an “OFF” status of the system.

[0068] In some embodiments, the system, kit, or platform comprises a plurality of analyte binding molecules. In some embodiments, each analyte binding molecule has a different binding moiety, which binds to a different analyte. The invader sequence may be the same or different for each analyte binding molecule. The toe sequence may be the same or different for each analyte binding molecule. In some embodiments, each detection probe comprises a different dye-acceptor pair.

[0069] In some embodiments, the system is assembled into a kit. In embodiments, the kit comprises, in separated components, (a) the analyte binding molecule, (b) the detection probe, and (c) the acceptor fragment.

[0070] In an embodiment, provided herein is a method of using the system, kit, or platform for detecting an analyte in a sample. In some embodiments, the method comprises

[0071] adding to the sample the analyte binding molecule and the nucleic acid detection probe of the fifth configuration;

[0072] incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and

[0073] detecting the signal.

[0074] In some embodiments, the method further comprises adding to the sample the acceptor fragment; and optionally, detecting the signal.

[0075] As used herein, the term “analyte binding moiety” refers to a protein that specifically binds to an analyte. In some embodiments, the analyte binding molecule is a protein or a peptide. In some embodiments, the analyte binding moiety is an antibody. The terms “analyte,” “marker”, and “target molecule” are used interchangeable to refer to an antigen or other nucleic acid and / or peptide molecule.

[0076] In embodiments, the analyte binding moiety is linked to the invader molecule via a linker. Suitable linkers include, without limitation, crosslinking agents having reactive moieties specific to various functional groups (e.g., sulfhydryls, amines, carbohydrates, azide, and alkyne). Maleimide, haloacetyl, pyridyl disulfide, (methyl)-tetrazine, and trans-cyclooctene (TCO) linkers may be used.

[0077] The terms “toe” or “toehold” are used interchangeably to refer to a single-stranded region of a nucleic acid molecule that initiates the strand displacement process during toehold- mediated strand displacement. In some embodiments, the toehold sequence is up to 20 nucleotides in length; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 nucleotides.

[0078] The terms “dye molecule,” “fluorophore” and “fluorochrome” are used interchangeably to refer to a fluorescent molecule. Suitable dye molecules include, without limitation, 6-FAM™, JOE™, Cy5®, Cy3®, Alexa Fluor dyes and Atto-Tec Dyes, such as Atto390, Atto425, Atto488, Atto550, Atto647N.

[0079] The term “acceptor molecule” refers to a molecule that decreases the fluorescence intensity of a dye molecule when the two molecules are in proximity to each other. In some embodiments, the acceptor molecule is a second dye molecule which emits a different signal than the first dye molecule via fluorescence resonance energy transfer (FRET) when the two molecules are in proximity to each other. Suitable acceptor molecules include, without limitation, dabcyl, TAMRA™, BHQ™-1, -2 or -3, and BMN-Q620.

[0080] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds. Nucleic acids include deoxyribonucleic acids (DNA); ribonucleic acids (RNA) such as messenger RNA (mRNA), etc; peptide nucleic acids (PNA); locked nucleic acids (LNA); etc. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or include non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3 ' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxy thy mi dine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2 '-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0081] Nucleic acids, proteins, and / or other compositions described herein may be purified. As used herein, “purified” means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, spectrophotometer, etc.

[0082] As used herein, the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “5 -C-A-G- T,” is complementary to the sequence “5'-A-C-T-G ” Complementarity can be “partial” or “total.” “Partial” complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. “Total” or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.

[0083] As used herein, the term “specific to” is used to define the relationship between macromolecular binding partners. For example, as used above, two nucleotide sequences that possess total complementarity to one another would be considered “specific” for one another, i.e., each totally complementary nucleotide would be specific to the other.

[0084] Methods of making polynucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solidphase synthesis methods are preferred for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am.Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124: 13684-13685 (2002).

[0085] The term “hybridization,” as used herein, refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning- A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).

[0086] As used herein, “fixation” or “fixing” refers to the process of chemically stabilizing organic, inorganic, or a combination of organic and inorganic molecules through the use of reagents, known as “fixatives”. Exemplary fixatives for the present disclosure include, but are not limited to, formaldehyde, formaldehyde derived from paraformaldehyde, formalin, phosphate buffered formalin, formal calcium, formal saline, zinc formalin, alcoholic formalin, glutaraldehyde, other organic aldehydes, methanol, ethanol, isopropanol, or other organic alcohols, or solutions containing organic alcohols or aldehydes.

[0087] As used herein, the terms “proteins,” “peptides,” and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms“protein” and “polypeptide” refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to an encoded gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The antibodies of the present invention are polypeptides, as well the antigen-binding fragments and fragments thereof.

[0088] The terms "antibody" or "antibody molecule" are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain. The term "antibody" or "antibody molecule" as used herein is thus intended to include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibodies, nanobodies, antigen-binding fragments, and genetically engineered antibodies, among others, as long as the characteristic properties (e.g., ability to bind CD30) are retained. The term "antibody fragment" as used herein is intended to include any appropriate antibody fragment that displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments.

[0089] As stated above, the term "antibody" includes "antibody fragments" or "antibody- derived fragments" and "antigen binding fragments" which comprise an antigen binding domain. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), (see for instance Bird et al. , Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context.

[0090] Antibodies can be genetically engineered from the CDRs and monoclonal antibody sequences described herein into antibodies and antibody fragments by using conventional techniques such as, for example, synthesis by recombinant techniques or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.

[0091] The antibodies or antibody fragments can be wholly or partially synthetically produced. Thus the antibody may be from any appropriate source, for example recombinant sources and / or produced in transgenic animals or transgenic plants. Thus, the antibody molecules can be produced in vitro or in vivo. The antibody or antibody fragment can be made that comprises all or a portion of a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, IgM or IgD constant region.

[0092] Furthermore, the antibody or antibody fragment can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions may be produced wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art.

[0093] The term "fragment" as used herein refers to fragments of biological relevance (functional fragment), e.g., fragments which can contribute to or enable antigen binding, e.g., form part or all of the antigen binding site or can contribute to the prevention of the antigen interacting with its natural ligands. Fragments in some embodiments comprise a heavy chain variable region (VH domain) and light chain variable region (VL) of the disclosure. In some embodiments, the fragments comprise one or more of the heavy chain complementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site.

[0094] Protein and nucleic acid sequence identities are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Set. USA 87: 2267-2268; Altschul etal., 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula (Karlin and Altschul, 1990), the disclosure of which is incorporated by reference in its entirety. The BLAST programs can be used with the default parameters or with modified parameters provided by the user.

[0095] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0096] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 85% sequence identity to the SEQ ID. Alternatively, percent identity can be any integer from 85% to 100%. More preferred embodiments include at least: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0097] " Substantial identity" of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 85%. Preferred percent identity of polypeptides can be any integer from 85% to 100%. More preferred embodiments include at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0098] In some embodiments, the isolated antibody or fragment thereof is directly or indirectly linked to a tag or agent. In some embodiments, the antibody or fragment thereof is conjugated tothe tag or agent. In other embodiments, the tag or agent is a polypeptide, wherein the polypeptide is translated concurrently with the antibody polypeptide sequence.

[0099] The kits provided herein include the systems for detection of analytes. A kit may further include buffers for antibody dilution and / or sample washing. A kit may further comprise instructions for use, such as, e.g. a publication, a recording, a diagram, or any other medium of expression which is used to communicate the usefulness of the reagents (e.g. analyte binding molecule, detection reagent, acceptor fragment, acceptor strand, dye strand, invader strand, etc). The instructional material of the kit can, for example, be affixed to a container which contains the reagents, or be shipped together with a container which contains the reagents. A kit may further include a quality control certificate.

[0100] As used herein "sample" or "biological sample" refers to a sample taken from a subject, such as but not limited to, a blood, tissue, or bodily fluid sample. In embodiments, the sample is a fixed sample. In some embodiments, the sample is a fixed-formalin paraffin-embedded sample. In embodiments, the sample is not fixed. In embodiments, the sample includes live cells or tissue such as human-, animal-, or plant-derived tissue or cells.

[0101] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0102] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0103] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to thosecomponents recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0104] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0105] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0106] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0107] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspectof a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0108] For the purposes of this disclosure and the appended claims, the use of the terms "substantially", "approximately", "about" and similar terms in reference to a descriptor of a value, element, property or characteristic at hand is intended to emphasize that the value, element, property, or characteristic referred to, while not necessarily being exactly as stated, would nevertheless be considered, for practical purposes, as stated by a person of skill in the art. These terms, as applied to a specified characteristic or quality descriptor means "mostly", "mainly", "considerably", "by and large", "essentially", "to great or significant extent", "largely but not necessarily wholly the same" such as to reasonably denote language of approximation and describe the specified characteristic or descriptor so that its scope would be understood by a person of ordinary skill in the art. In one specific case, the terms "approximately", "substantially", and "about", when used in reference to a numerical value, represent a range of plus or minus 20% with respect to the specified value, more preferably plus or minus 10%, even more preferably plus or minus 5%, most preferably plus or minus 2% with respect to the specified value. As a non-limiting example, two values being "substantially equal" to one another implies that the difference between the two values may be within the range of + / - 20% of the value itself, preferably within the + / - 10% range of the value itself, more preferably within the range of + / - 5% of the value itself, and even more preferably within the range of + / - 2% or less of the value itself. The use of these terms in describing a chosen characteristic or concept neither implies nor provides any basis for indefiniteness and for adding a numerical limitation to the specified characteristic or descriptor. As understood by a skilled artisan, the practical deviation of the exact value or characteristic of such value, element, or property from that stated falls and may vary within a numerical range defined by an experimental measurement error that is typical when using a measurement method accepted in the art for such purposes.

[0109] The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.EXAMPLES

[0110] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0111] Example 1. Testing the system as shown in Figure 3.

[0112] The system of Figure 3 was tested (without antibody), by evaluating fluorescent signal over time.

[0113] Fluorescence measurements were done in PBS on a fluorescent plate reader over a time of 3 min with measurements taken every 20 sec. After each addition of a new oligo, the sample was mixed, and fluorescent signal measurement began 30 sec after addition of the oligo. Oligos were added in the following order and concentration. 1. SeqlA (10 pM). 2. SeqlC (20 pM). 3. SeqlE (10 pM). 4. SeqlD (20 pM). For better visualization via a blacklight lamp, the samples were prepared as described above. Results as shown in Figure 10.

[0114] Example 2: The disclosed systems provide clean “off-on” switching capabilities.

[0115] An invader strand linked to an antibody targeting CD3 was contacted with human PBMCs (Figure 11 A). A dye-acceptor strand was then added, and cells were incubated for an additional 5 minutes (Figure 1 IB).

[0116] Human PBMCs were loaded onto ZellSafe chips and measurements were done on a ZellScanner ONE. For the non-supressed probe, 100 nM of SeqlA in PBS was added to the chip, incubated for 5 min at room temperature and washed with 10ml of PBS. For the suppressed probe, SeqlA and SeqlC were hybridized first. In short, the oligos were added to hybridization buffer (10 mM HEPES pH 7.4, 50 mM NaCl) for a concentration of 1 pM of SeqlA and 2 pM for SeqlC. The sample was incubated at 95 °C for 5 min before cooling to room temperature. The hybridized sample was diluted with PBS for a concentration of 100 nM for SeqlA, added to the chip, incubated for 5 min at room temperature and washed with 10 ml PBS. Results are shown in Figure 11.

[0117] Example 3: The disclosed systems provide clean “off-on” switching capabilities with multiple channels - testing the system as seen in Figure 3

[0118] Human PBMCs were loaded onto ZellSafe chips and measurements were done on a Zell Scanner ONE. PBMCs were incubated with antibodies targeting CD4 (clone RPA-T4) and CD45 (clone HI30) in PBS for 10 min followed by a washing step with 10 ml PBS. The antibody for CD4 was conjugated to an oligo with SeqlE and the antibody against CD45 with an oligo with SeqlF. To detect CD4, a detection probe consisting of SeqlA and SeqlC was added to the cells at a concentration of 100 nM in PBS and incubated for 5 min, followed by a washing step with 10 ml PBS. Afterwards fluorescent pictures were taken in the appropriate channels. Next, CD45 was detected by adding a detection probe consisting of Seq IB and Seq 1C as described before. To suppress the signal of both the CD4 and CD45 antibody, SeqlD was added at a concentration of 200 nM in PBS for 5 min followed by a washing step with 10 ml PBS. Results are shown in Figure 12.

[0119] Example 4: The disclosed systems provide clean “off-on” switching capabilities with multiple channels - testing the system as seen in Figure 9

[0120] Human PBMCs were loaded onto ZellSafe chips and measurements were done on a Zell Scanner ONE. PBMCs were incubated with antibodies targeting CD4 (clone RPA-T4) and CD45 (clone HI30) in PBS for 10 min followed by a washing step with 10 ml PBS. The antibody for CD4 was conjugated to an oligo with Seq2C and the antibody against CD45 with an oligo with Seq2D. To detect CD4, an oligo with Seq2A was added at a concentration of 100 nM in PBS and incubated for 5 min, followed by a washing step with 10 ml PBS. Afterwards fluorescent pictures were taken in the appropriate channels. Next, CD45 was detected by adding an oligo with Seq2B as described before. To suppress the signal of both the CD4 and CD45 antibody, Seq2E was added at a concentration of 200 nM in PBS for 5 min followed by a washing step with 10 ml PB S. Results are shown in Figure 13.

[0121] Example 5: Conjugation of oligos to antibodies

[0122] All antibodies were used at a concentration of 1 mg / ml in PBS + 0.03% sodium azide. Stock solutions of oligos were 100 pM in dH2O. First, 1 M NaHCCh was added in a ratio of 1: 10to the antibody solution. Next, the antibody solution was incubated with a 15x molar excess of NHS-PEG2-methyl tetrazine for 1 h at room temperature and cleaned via a micro spin desalting column (7K cutoff). A trans-cycloocten (TCO) modified oligo is added at a 2x molar excess to the antibody solution and incubated for 1 h at room temperature. Afterwards, the sample is separated via anion exchange chromatography and peaks of oligo conjugated antibodies were pooled followed by a buffer exchange to PBS + 1 mM EDTA and a concentration step via amicon spin columns (50K cutoff).

[0123] The sequences of the nucleic acids used in FIGs. 10-13 are provided in Table 1.

[0124] Table 1.

[0125] In Table 1 , the bold sequences are the toe region; underlined sequences are the overhang sequence.

[0126] Table 2. Additional exemplary sequences of nucleic acid strands useful in the present technology (FIG. 8) are provided. The dye sequence is bolded. The analyte target region is underlined. The acceptor sequence is italicized. The toehold-region is bolded and underlined.

[0127] Table 2.

Claims

CLAIMS1. A system, kit, or platform comprising: a. at least one analyte binding molecule comprising: i) an analyte binding moiety; and ii) a nucleic acid invader molecule comprising: a) a toehold region; and b) a main region; wherein the analyte binding moiety is linked to the invader molecule; b. a nucleic acid detection reagent comprising: i) a dye strand comprising a) a dye sequence that is complementary to the toehold region and the main region of the invader molecule, and b) a dye molecule of a dye-acceptor pair; ii) an acceptor strand comprising a) an acceptor sequence that comprises the main region, and b) an acceptor molecule of the dye-acceptor pair; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule.

2. The system, kit, or platform of claim 1 or 2, wherein the analyte binding moiety is a protein or a peptide.

3. The system, kit, or platform of any one of claims 1-3, wherein the analyte binding moiety is an antibody.

4. The system, kit, or platform of any one of claims 1-4, wherein at least the toehold region and the main region of the invader molecule are single-stranded.

5. The system, kit, or platform of any one of claims 1-4, wherein the dye strand and the acceptor strand are separate nucleic acid strands.

6. The system, kit, or platform of any one of claims 1-4, wherein the dye strand and the acceptor strand are a single nucleic acid strand, and wherein the single nucleic acid strand forms a secondary structure when the dye sequence and the acceptor sequence hybridize to each other.

7. The system, kit, or platform of any one of claims 1-6, wherein the toe sequence is up to 20 nucleotides in length.

8. The system, kit, or platform of any one of claims 1-7, wherein the acceptor molecule is a second dye molecule, and wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule emits a second signal.

9. The system, kit, or platform of any one of claims 1-8, wherein the dye sequence further comprises a single-stranded overhang sequence in proximity to the dye molecule; wherein the system further comprises an acceptor fragment that comprises the acceptor molecule and a sequence that is complementary to the overhang sequence; and wherein when the acceptor fragment hybridizes to the overhang sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

10. The system, kit, or platform of any one of claims 1-8, wherein the invader molecule further comprises a single-stranded overhang sequence; wherein the system further comprises an acceptor fragment that comprises the acceptor molecule and a sequence that is complementary to the overhang sequence; and wherein when the acceptor fragment hybridizes to the overhang sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

11. The system, kit, or platform of any one of claims 1-10, wherein the dye sequence, the acceptor sequence, and the invader molecule are selected from DNA, LNA, PNA, and RNA.

12. The system, kit, or platform of any one of claims 1-11, wherein the dye molecule is a fluorophore.

13. The system, kit, or platform of any one of claims 1-12, comprising a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

14. The system, kit, or platform of claim 13, wherein each of the plurality of analyte binding molecules comprises the same invader molecule.

15. The system, kit, or platform of claim 13, wherein each of the plurality of analyte binding molecules comprises a different invader molecule, and wherein the system, kit, or platform further comprises a plurality of detection reagents, wherein the dye sequence and acceptor sequence of each detection reagent correspond to one of the invader molecules.

16. The system, kit, or platform of claim 13-15, wherein each detection reagent comprises a different dye-acceptor pair or the same dye-acceptor pair.

17. A method for detecting at least one analyte in a sample, the method comprising: adding to the sample a. at least one analyte binding molecule comprising: i) an analyte binding moiety; and ii) a nucleic acid invader molecule comprising: a) a toehold region; and b) a main region; wherein the analyte binding moiety is linked to the invader molecule; b. a nucleic acid detection reagent comprising: i) a dye strand comprising a) a dye sequence that is complementary to the toehold region and the main region of the invader molecule, and b) a dye molecule of a dye-acceptor pair;ii) an acceptor strand comprising a) an acceptor sequence that comprises the main region, and b) an acceptor molecule of the dye-acceptor pair; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

18. The method of claim 17, wherein the analyte binding moiety is a protein or a peptide.

19. The method of claim 17 or 18, wherein the analyte binding moiety is an antibody.

20. The method of any one of claims 17-19, wherein at least the toehold region and the main region of the invader molecule are single-stranded.

21. The method of any one of claims 17-20, wherein the dye strand and the acceptor strand are separate nucleic acid strands.

22. The method of any one of claims 17-20, wherein the dye strand and the acceptor strand are a single nucleic acid strand, and wherein the single nucleic acid strand forms a secondary structure when the dye sequence and the acceptor sequence hybridize to each other.

23. The method of any one of claims 17-22, wherein the toehold region is up to 20 nucleotides in length.

24. The method of any one of claims 17-23, wherein the acceptor molecule is a second dye molecule, wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule emits a second signal, and wherein the method further comprises detecting the second signal.

25. The method of any one of claims 17-24, wherein the dye sequence further comprises a single-stranded overhang sequence in proximity to the dye molecule; and wherein the method further comprises: adding to the sample an acceptor fragment that comprises the acceptor molecule and a sequence that is complementary to the overhang sequence; wherein when the acceptor fragment hybridizes to the overhang sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

26. The method of any one of claims 17-25, wherein the dye sequence, the acceptor sequence, and the invader molecule are selected from DNA, LNA, PNA, and RNA.

27. The method of any one of claims 17-26, wherein the dye molecule is a fluorophore.

28. The method of any one of claims 17-27, wherein the at least one analyte binding molecule comprises a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

29. The method of claim 28, wherein each of the plurality of analyte binding molecules comprises the same invader molecule.

30. The method of claim 28, wherein each of the plurality of analyte binding molecules comprises a different invader molecule, and wherein the method further comprises adding to the sample a plurality of detection reagents, wherein the dye sequence and acceptor sequence of each detection reagent correspond to one of the invader molecules.

31. The method of claim 30, wherein each detection reagent comprises a different dyeacceptor pair or the same dye-acceptor pair.

32. A system, kit, or platform comprising: a. at least one analyte binding molecule comprising: i) an analyte binding moiety linked to a nucleic acid invader molecule comprising a target sequence; b. a nucleic acid detection reagent comprising: i) a dye sequence on a first end of the detection reagent;ii) a dye molecule of a dye-acceptor pair linked to the dye sequence; iii) an acceptor sequence on a second end of the detection reagent; iv) an acceptor molecule of the dye-acceptor pair linked to the acceptor sequence; and v) a detection sequence between the dye sequence and the acceptor sequence; wherein the dye sequence and the acceptor sequence are complementary to each other; wherein the detection sequence is complementary to the target sequence; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; and wherein when the detection sequence hybridizes to the target sequence, the acceptor molecule is unable to suppress the signal emitted by the dye molecule.

33. The system, kit, or platform of claim 32, wherein the analyte binding moiety is a protein or a peptide.

34. The system, kit, or platform of claim 32 or 33, wherein the analyte binding moiety is an antibody.

35. The system, kit, or platform of any one of claims 32-34, wherein the acceptor molecule is a second dye molecule, and wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule emits a second signal.

36. The system, kit, or platform of any one of claims 32-35, further comprising an acceptor fragment that comprises the acceptor molecule and a sequence that is complementary to the dye sequence; wherein when the acceptor fragment hybridizes to the dye sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

37. The system, kit, or platform of any one of claims 32-36, wherein the dye sequence, the acceptor sequence, and the target sequence are selected from DNA, LNA, PNA, and RNA.

38. The system, kit, or platform of any one of claims 32-37, wherein the dye molecule is a fluorophore.

39. The system, kit, or platform of any one of claims 32-38, comprising a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

40. The system, kit, or platform of claim 39, wherein each of the plurality of analyte binding molecules comprises the same target sequence.

41. The system, kit, or platform of claim 39, wherein each of the plurality of analyte binding molecules comprises a different target sequence, and wherein the system, kit, or platform further comprises a plurality of detection reagents, wherein each detection sequence corresponds to one of the target sequences.

42. The system, kit, or platform of claim 41, wherein each detection reagent comprises a different dye-acceptor pair or the same dye-acceptor pair.

43. A method for detecting at least one analyte in a sample, the method comprising: adding to the sample a. at least one analyte binding molecule comprising: i) an analyte binding moiety linked to a nucleic acid invader molecule comprising a target sequence; b. a nucleic acid detection reagent comprising: i) a dye sequence on a first end of the detection reagent; ii) a dye molecule of a dye-acceptor pair linked to the dye sequence; iii) an acceptor sequence on a second end of the detection reagent; iv) an acceptor molecule of the dye-acceptor pair linked to the acceptor sequence; and v) a detection sequence between the dye sequence and the acceptor sequence;wherein the dye sequence and the acceptor sequence are complementary to each other; wherein the detection sequence is complementary to the target sequence; wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; and wherein when the detection sequence hybridizes to the target sequence, the acceptor molecule is unable to suppress the signal emitted by the dye molecule; incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

44. The method of claim 43, wherein the analyte binding moiety is a protein or a peptide.

45. The method of claim 43 or 44, wherein the analyte binding moiety is an antibody.

46. The method of any one of claims 43-45, wherein the acceptor molecule is a second dye molecule, wherein when the dye sequence and the acceptor sequence hybridize to each other, the acceptor molecule emits a second signal, and wherein the method further comprises detecting the second signal.

47. The method of any one of claims 43-46, wherein the dye sequence, the acceptor sequence, the detection sequence and the target sequence are selected from DNA, LNA, PNA, and RNA.

48. The method of any one of claims 43-47, wherein the dye molecule is a fluorophore.

49. The method of any one of claims 43-48, wherein the at least one analyte binding molecule comprises a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

50. The method of claim 49, wherein each of the plurality of analyte binding molecules comprises the same target sequence.

51. The method of claim 49, wherein each of the plurality of analyte binding molecules comprises a different target sequence, and wherein the method further comprises adding to the sample a plurality of detection reagents, wherein the detection sequence of each detection reagent corresponds to one of the target sequences.

52. The method of claim 51, wherein each detection reagent comprises a different dyeacceptor pair or the same dye-acceptor pair.

53. A system, kit, or platform comprising: a. at least one analyte binding molecule comprising: i) an analyte binding moiety; and ii) a nucleic acid analyte strand comprising: a) a dye target region; and b) an acceptor target region; wherein the analyte binding moiety is linked to the analyte strand; b. a nucleic acid dye strand comprising: i) a dye sequence that is complementary to the dye target region, and ii) a dye molecule of a dye-acceptor pair; c. a nucleic acid acceptor strand comprising: i) an acceptor sequence that is complementary to the acceptor target region; ii) a toehold sequence; and iii) an acceptor molecule of the dye-acceptor pair; and d. a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; wherein when the dye sequence and the acceptor sequence hybridize to the analyte strand, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; andwherein when the invader molecule hybridizes to the acceptor strand, the acceptor molecule is unable to suppress the signal.

54. The system, kit, or platform of claim 53, wherein the analyte binding moiety is a protein or a peptide.

55. The system, kit, or platform of claim 53 or 54, wherein the analyte binding moiety is an antibody.

56. The system, kit, or platform of any one of claims 53-55, wherein the toehold region is up to 20 nucleotides in length.

57. The system, kit, or platform of any one of claims 53-56, wherein the acceptor molecule is a second dye molecule, and wherein when the dye sequence and the acceptor sequence hybridize to the analyte strand, the acceptor molecule emits a second signal.

58. The system, kit, or platform of any one of claims 53-57, further comprising an acceptor fragment that comprises the acceptor sequence and the acceptor molecule, wherein when the acceptor fragment hybridizes to the acceptor target region, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

59. The system, kit, or platform of any one of claims 53-58, wherein the analyte strand, the dye sequence, the acceptor sequence, the toehold sequence, and the invader molecule are selected from DNA, LNA, PNA, and RNA.

60. The system, kit, or platform of any one of claims 53-59, wherein the dye molecule is a fluorophore.

61. The system, kit, or platform of any one of claims 53-59, wherein the dye strand further comprises a second toehold sequence, and wherein the system further comprises e. a nucleic acid second invader molecule comprising: i) a second invader sequence that is complementary to the dye sequence and the second toehold sequence; and ii) the acceptor molecule of the dye-acceptor pair;wherein when the second invader sequence hybridizes to the dye sequence and the toe sequence, the acceptor molecule on the second invader molecule suppresses the signal emitted by the dye molecule.

62. The system, kit, or platform of any one of claims 53-61, wherein the at least one analyte binding molecule comprises a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

63. The system, kit, or platform of claim 62, further comprising: a plurality of acceptor strands, wherein each acceptor strand corresponds to a different analyte binding molecule; and a plurality of invader molecules, wherein each invader molecule corresponds to one of the acceptor strands.

64. The system, kit, or platform of claim 62 or 63, further comprising: a plurality of a plurality of dye strands, wherein each dye strand corresponds to a different analyte binding molecule; and a plurality of second invader molecules, wherein each second invader molecule corresponds to one of the dye strands.

65. The system, kit, or platform of any one of claims 62-64, wherein each dye strand and acceptor strand that correspond to one analyte binding molecule comprise a different dye-acceptor pair or the same dye-acceptor pair.

66. A method for detecting at least one analyte in a sample, the method comprising: adding to the sample a. at least one analyte binding molecule comprising: i) an analyte binding moiety; and ii) a nucleic acid analyte strand comprising: a) a dye target region; and b) an acceptor target region;wherein the analyte binding moiety is linked to the analyte strand; b. a nucleic acid dye strand comprising: i) a dye sequence that is complementary to the dye target region, and ii) a dye molecule of a dye-acceptor pair; c. a nucleic acid acceptor strand comprising: i) an acceptor sequence that is complementary to the acceptor target region; ii) a toehold sequence; and iii) an acceptor molecule of the dye-acceptor pair; wherein when the dye sequence and the acceptor sequence hybridize to the analyte strand, the acceptor molecule is positioned to suppress a signal emitted by the dye molecule; incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

67. The method of claim 66, wherein the analyte binding moiety is a protein or a peptide.

68. The method of claim 66 or 67, wherein the analyte binding moiety is an antibody.

69. The method of any one of claims 66-68, wherein the toehold region is up to 20 nucleotides in length.

70. The method of any one of claims 66-69, wherein the acceptor molecule is a second dye molecule, wherein when the dye sequence and the acceptor sequence hybridize to the nucleic acid analyte strand, the acceptor molecule emits a second signal, and wherein the method further comprises detecting the second signal.

71. The method of any one of claims 66-69, further comprising: adding to the sample a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; anddetecting the signal; wherein when the invader molecule hybridizes to the acceptor strand and the toehold sequence, the acceptor molecule is unable to suppress the signal.

72. The method of claim 71, further comprising: adding to the sample an acceptor fragment that comprises the acceptor molecule and a sequence that is complementary to the acceptor target region; and detecting the signal; wherein when the acceptor fragment hybridizes to the acceptor target region, the acceptor molecule suppresses the signal emitted by the dye molecule.

73. The method of any one of claims 66-72, wherein the dye target region, the acceptor target region, the dye strand, the acceptor strand, and the invader molecule are selected from DNA, LNA, PNA, and RNA.

74. The method of any one of claims 66-73, wherein the dye molecule is a fluorophore.

75. The method of any one of claims 66-74, wherein the dye strand further comprises a second toehold sequence, and wherein the method further comprises adding to the system a nucleic acid second invader molecule comprising: i) a second invader sequence that is complementary to the dye sequence and the second toehold sequence; and ii) the acceptor molecule of the dye-acceptor pair; wherein when the second invader sequence hybridizes to the dye sequence and the toe sequence, the acceptor molecule on the second invader molecule suppresses the signal emitted by the dye molecule; and detecting the signal.

76. The method of any one of claims 66-75, wherein the at least one analyte binding molecule comprises a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

77. The method of claim 76, further comprising adding to the sample: a plurality of acceptor strands, wherein each acceptor strand corresponds to a different analyte binding molecule; and a plurality of invader molecules, wherein each invader molecule corresponds to one of the acceptor strands.

78. The method of claim 76, further comprising adding to the sample: a plurality of a plurality of dye strands, wherein each dye strand corresponds to a different analyte binding molecule; and a plurality of second invader molecules, wherein each second invader molecule corresponds to one of the dye strands.

79. The method of any one of claims 76-78, wherein each dye strand and acceptor strand that correspond to one analyte binding molecule comprise a different dye-acceptor pair or the same dye-acceptor pair.

80. A system, kit, or platform comprising: a. at least one analyte binding molecule comprising: i) an analyte binding moiety; and ii) a nucleic acid analyte strand; wherein the analyte binding moiety is linked to the analyte strand; b. a nucleic acid dye strand comprising: i) an analyte target region that is complementary to the analyte strand; ii) a dye sequence; and iii) a dye molecule of a dye-acceptor pair; and c. a nucleic acid acceptor strand comprising:i) an acceptor sequence that is complementary to the dye sequence; ii) a toehold sequence; and iii) an acceptor molecule of the dye-acceptor pair; and d. a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; wherein when the analyte target region hybridizes to the analyte strand, a signal is emitted by the dye molecule; wherein when the acceptor strand hybridizes to the dye sequence, the acceptor molecule is positioned to suppress the signal; and wherein when the invader molecule hybridizes to the acceptor strand, the signal is emitted.

81. The system, kit, or platform of claim 80, wherein the analyte binding moiety is a protein or a peptide.

82. The system, kit, or platform of claim 80 or 81, wherein the analyte binding moiety is an antibody.

83. The system, kit, or platform of any one of claims 80-82, wherein he toehold region is up to 20 nucleotides in length.

84. The system, kit, or platform of any one of claims 80-82, wherein the acceptor molecule is a second dye molecule, and wherein when the acceptor strand hybridizes to the dye sequence, the acceptor molecule emits a second signal.

85. The system, kit, or platform of any one of claims 80-83, further comprising an acceptor fragment that comprises the acceptor sequence and the acceptor molecule, wherein when the acceptor fragment hybridizes to the dye sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

86. The system, kit, or platform of any one of claims 80-84, wherein the analyte strand, the dye sequence, the acceptor sequence, the toehold sequence, and the invader molecule are selected from DNA, LNA, PNA, and RNA.

87. The system, kit, or platform of any one of claims 80-85, wherein the dye molecule is a fluorophore.

88. The system, kit, or platform of any one of claims 80-86, wherein the at least one analyte binding molecule comprises a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

89. The system, kit, or platform of claim 87, further comprising: a plurality of acceptor strands; and a plurality of dye strands; a plurality of invader molecules; and a plurality of acceptor fragments; wherein each acceptor strand and dye strand correspond to one of the analyte binding molecules; wherein each invader molecule corresponds to one acceptor strands; and wherein each acceptor fragment corresponds to one of the dye strands.

90. The system, kit, or platform of claim 89, wherein each dye strand and acceptor strand comprise a different dye-acceptor pair or the same dye-acceptor pair.

91. A method for detecting at least one analyte in a sample, the method comprising: adding to the sample a. at least one analyte binding molecule comprising: i) an analyte binding moiety; and ii) a nucleic acid analyte strand; wherein the analyte binding moiety is linked to the analyte strand; b. a nucleic acid dye strand comprising: i) an analyte target region that is complementary to the analyte strand; ii) a dye sequence; andiii) a dye molecule of a dye-acceptor pair; and c. a nucleic acid acceptor strand comprising: i) an acceptor sequence that is complementary to the dye sequence; ii) a toehold sequence; and iii) an acceptor molecule of the dye-acceptor pair; and wherein when the analyte target region hybridizes to the analyte strand, a signal is emitted by the dye molecule; wherein when the acceptor strand hybridizes to the dye sequence, the acceptor molecule is positioned to suppress the signal; and incubating the sample under conditions that promote binding of the analyte binding moiety to the analyte; and detecting the signal.

92. The method of claim 91, wherein the analyte binding moiety is a protein or a peptide.

93. The method of claim 91 or 92, wherein the analyte binding moiety is an antibody.

94. The method of any one of claims 91-93, wherein the toehold region is up to 20 nucleotides in length.

95. The method of any one of claims 91-94, wherein the acceptor molecule is a second dye molecule, and wherein when the acceptor strand hybridizes to the dye sequence, the acceptor molecule emits a second signal.

96. The method of any one of claims 91-95, further comprising: adding to the sample a nucleic acid invader molecule that is complementary to the acceptor sequence and the toehold sequence; detecting the signal; wherein when the invader molecule hybridizes to the acceptor strand, the signal is emitted.

97. The method of claim 98, further comprising:adding to the sample an acceptor fragment that comprises the acceptor sequence and the acceptor molecule, and detecting the signal; wherein when the acceptor fragment hybridizes to the dye sequence, the acceptor molecule on the acceptor fragment suppresses the signal emitted by the dye molecule.

98. The method of any one of claims 91-97, wherein the analyte strand, the dye sequence, the acceptor sequence, the toehold sequence, and the invader molecule are selected from DNA, LNA, PNA, and RNA.

99. The method of any one of claims 91-98, wherein the dye molecule is a fluorophore.

100. The method of any one of claims 91-100, wherein the at least one analyte binding molecule comprises a plurality of analyte binding molecules, wherein each analyte binding moiety binds to a different analyte.

101. The method of claim 100, further comprising adding to the sample: a plurality of acceptor strands; a plurality of dye strands; a plurality of invader molecules; and a plurality of acceptor fragments; wherein each acceptor strand and dye strand correspond to one of the analyte binding molecules; wherein each invader molecule corresponds to one acceptor strands; and wherein each acceptor fragment corresponds to one of the dye strands.

102. The method of claim 101, wherein each dye strand and acceptor strand comprise a different dye-acceptor pair or the same dye-acceptor pair.

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