Oligomer-induced reconstitution of bioluminescence sensors for multiplex detection of analytes

Abstract

Described herein are fusion proteins and methods of using those fusion proteins for the detection and identification of analytes (e.g., multimeric proteins expressed by pathogenic organisms or mammalian cells). In some embodiments, it includes a plurality of pairs of fusion proteins distinct for one analyte wherein each pair comprises i) a first fusion protein comprising one or more binding domains fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding domains fused to a second portion of the same reporter protein, wherein the first and second portions of the reporter protein, when together, comprise the reporter protein in its entirety and restore its function, wherein the first fusion protein and / or the second fusion protein further comprises a fluorophore, and wherein the fluorophore for each pair has a distinguishable emission spectrum.

Description

[0001]Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Oligomer-induced Reconstitution of Bioluminescence Sensors for Multiplex Detection of Analytes RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 696,301, filed on September 18, 2024, the entire contents of which is hereby incorporated by reference in their entirety. REFERNECE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (B080170396WO00-SEQ-EAS.xml; Size: 163,752 bytes; and Date of Creation: September 17, 2025) is herein incorporated by reference in their entirety. TECHNICAL FIELD The present disclosure generally relates to fusion proteins for detection of protein analytes in samples. BACKGROUND Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) is employed as the gold standard for the confirmatory diagnosis of infectious diseases by amplifying and identifying the nucleic acids from pathogens with high sensitivity and specificity yet it is not readily adopted as the prevalent method for home diagnostics due to the requirement of sample transportation to laboratories, complicated sample preparation, skilled technicians, expensive equipment, and long turnaround time (12h - 72h). There is a need for rapid diagnostic methods and kits for the detection and identification of infection diseases. SUMMARY Provided herein are methods for detecting and identifying the presence or absence of one or more analytes, e.g., multimeric analytes, optionally multimeric proteins expressed by pathogenic organisms, in a sample. The methods comprise: a) providing a sample, optionally a sample from a subject or an environmental sample; 1#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 b) contacting the sample with a plurality of pairs of fusion proteins wherein each pair of fusion protein is distinct for one analyte and each pair comprises i) a first fusion protein comprising one or more binding domains fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding domains that bind to the same analyte as the first fusion protein fused to a second portion of the same reporter protein as the first fusion protein, wherein the first and second portions of the reporter protein, when together, comprise the reporter protein in its entirety and restore its function, wherein the first fusion protein and / or the second fusion protein further comprises a fluorophore (preferably, wherein only one of the fusion proteins comprises a the fluorophore), and wherein the fluorophore for each pair has a distinguishable (i.e., non-overlapping) emission spectrum; and c) detecting and / or identifying one or more analytes based on the emissions from one or more of the fluorophores, optionally wherein detection occurs within 3 minutes or within 30-60 seconds. In some embodiments, the presence of two or more analytes is based on detection of a combination of distinguishable emissions from two or more fluorophores. In some embodiments, the presence of two or more analytes is based on detection of distinguishable emissions from two or more fluorophores. In some embodiments, the one or more binding domains for each pair of the plurality of fusion proteins is specific for a distinct analyte, thereby making each pair of fusion protein specific for one analyte, optionally wherein the one or more binding domains are binders that recognize and bind at or near the apex of the distinct multimeric analyte, optionally to non-overlapping epitopes at or near the apex of the distinct multimeric analyte. In some embodiments, the first and second fusion proteins are configured to bind to the analyte to bring the first and second reporter protein fragments together within around 50 Å of each other, optionally less than 45 Å, less than 40 Å, less than 35 Å, less than 30 Å, less than 25 Å, less than 20 Å, less than 15 Å, less than 10 Å, less than 5 Å, or less than 1 Å, such as, about 45 Å to about 50 Å, about 40 Å to about 45 Å, about 35 Å to about 40 Å, about 30 Å to about 35 Å, about 25 Å to about 30 Å, about 20 Å to about 25 Å, about 15 Å to about 20 Å, about 10 Å to about 15 Å, about 5 Å to about 10 Å, about 1 Å to about 5 Å (e.g., about 50 Å, about 45 Å, about 40 Å, 2#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 about 35 Å, about 30 Å, about 25 Å, about 20 Å, about 15 Å, about 10 Å, about 5 Å, or about 1 Å). In some embodiments, the reporter protein is a luminescence-based enzyme. In some embodiments, the emission spectra of the reporter protein overlaps with the excitation spectra of the fluorophore. In some embodiments, the fluorophore is a small molecule fluorophore (or a plurality of small molecule fluorophores). In some embodiments, the fluorophore is fused either to the first fusion protein or the second fusion protein. In some embodiments, the fluorophore is different for each of the plurality of pairs of fusion proteins. In some embodiments, the reporter protein is luciferase. In some embodiments, the substrate for the reporter protein is luciferin or a derivative of luciferin. In some embodiments, the fluorophore is mNeonGreen, mCyRFP1, CyOFP1, and / or LSSmOrange. In some embodiments, step b) comprises contacting the sample with the plurality of pairs of fusion proteins under conditions effective for the reporter protein to generate a detectable signal. In some embodiments, conditions effective for the reporter protein to generate a detectable signal comprises the presence of a composition comprising a substrate for the reporter protein. In some embodiments, the first portion of the reporter protein and the second portion of the reporter protein are different sizes. In some embodiments, the fluorophore is genetically fused to the fusion protein comprising a smaller portion of the reporter protein. In some embodiments, the first portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO: 2. In some embodiments, the second portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO: 1. In some embodiments, the first portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO: 3. In some embodiments, the second portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO: 4. 3#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 In some embodiments, the first portion of the reporter protein is fused either to an N-terminal portion of the first fusion protein or to a C-terminal portion of the first fusion protein. In some embodiments, the second portion of the reporter protein is fused either to an N-terminal portion of the second fusion protein or to a C-terminal portion of the second fusion protein. In some embodiments, the first fusion protein further comprises a linker between the binding domain and the first portion of a reporter protein. In some embodiments, the second fusion protein further comprises a linker between the binding domain and the second portion of a reporter protein. In some embodiments, the first and second fusion proteins further comprise a purification tag. In some embodiments, the purification tag is a FLAG tag, a c-Myc tag, a biotin tag, a histidine tag, or combination thereof. In some embodiments, the first and / or second fusion proteins comprise one or more linkers between each component of the first and / or second fusion proteins. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids, e.g., 1-50 amino acids, long. In some embodiments, the one or more analytes are multimeric proteins expressed by one or more viruses, bacteria, fungus, parasites, or mammalian cells, optionally wherein the viruses, bacteria, fungus, or parasites are pathogenic. In some embodiments, the one or more pathogenic viruses comprises or consists of SARS-CoV-2, Influenza, and RSV. In some embodiments, at least one pair of fusion proteins comprises a pair of fusion proteins for SARS-CoV-2. In some embodiments, the pair of fusion proteins for SARS-CoV-2 is selected from the following: a) Histag-B3-nLuc(51-171) (SEQ ID NO: 27) and Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); b) Histag-B3-nLuc(51-171)-mNeonGreen (SEQ ID NO: 28) and Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); c) i) His-B3-L (SEQ ID NO: 53) or ii) His-L-B3 (SEQ ID NO: 54) and 4#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 i) Histag-B3-S-mNeonGreen (SEQ ID NO: 30) or ii) Histag-mNeonGreen-S- B3 (GSB3) (SEQ ID NO: 31); d) Histag-B3-L-mNeonGreen (SEQ ID NO: 32) and i) His-B3-S (SEQ ID NO: 51) or ii) His-S-B3 (SEQ ID NO: 52); e) His-L-I (SEQ ID NO: 56) and i) His-I-S-mNeonGreen (SEQ ID NO: 33), ii) His-I-mNeonGreen-S (SEQ ID NO: 34), or iii) His-Ihalf1-mNeonGreen-S- Ihalf2 (SEQ ID NO: 35). In some embodiments, at least one pair of fusion proteins comprises a pair of fusion proteins for Influenza. In some embodiments, the pair of fusion proteins for Influenza is selected from the following: i) His-84-L (SEQ ID NO: 74) or ii) L-84-His (SEQ ID NO: 75), and i) mCyRFP1-S-84-His (SEQ ID NO: 36), ii) S-mCyRFP1-84-His (SEQ ID NO: 37), iii) CyOFP1-S-84-His (SEQ ID NO: 38), iv) S-CyOFP1-84-His (SEQ ID NO: 39), v) LSSmOrange-S-84-His (SEQ ID NO: 40), or v) S- LSSmOrange-84-His (SEQ ID NO: 41). In some embodiments, at least one pair of fusion proteins comprises a pair of fusion proteins for RSV. In some embodiments, the pair of fusion proteins for RSV is selected from the following: i) His-MED-L (SEQ ID NO: 49) or ii) His-L-MED (SEQ ID NO: 48), and i) MED-S-LSSmOrange-Histag (SEQ ID NO: 42), ii) Histag- LSSmOrange-S-MED (SEQ ID NO: 43), iii) MED-S-mCyRFP1-Histag (SEQ ID NO: 44), iv) Histag-mCyRFP1-S-MED (SEQ ID NO: 45), v) MED-S-CyOFP1-Histag (SEQ ID NO: 46), or v) Histag-CyOFP1-S-MED (RSMED) (SEQ ID NO: 47). In some embodiments, the method is done in a single well. Also provided herein are systems or kits for detecting one or more analytes, e.g., multimeric analytes, in a sample, using a method described herein, optionally wherein the system comprises one or more of: A) a container for collecting a sample and B) a plurality of pairs of fusion proteins comprising pairs is that each bind to one of a plurality of analytes, wherein each pair comprises i) a first fusion protein comprising one or more binding domains that bind to a analyte fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding 5#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 domains that bind to the same analyte as the first fusion protein fused to a second portion of the same reporter protein as the first fusion protein; the plurality of pairs of fusion proteins can be provided in a single container. Where necessary, the systems or kits can also include a substrate for the reporter protein, e.g., in the same container or a separate container from the plurality of pairs of fusion proteins. In some embodiments, the one or more analytes are one or more multimeric proteins expressed by mammalian cells, or pathogenic viruses, parasites, fungus, or bacteria. In some embodiments, the one or more pathogenic viruses comprises or consists of SARS-CoV-2, Influenza, and RSV. In some embodiments, at least one pair of fusion proteins comprises a pair of fusion proteins for SARS-CoV-2. In some embodiments, the pair of fusion proteins for SARS-CoV-2 is selected from the following: a) Histag-B3-nLuc(51-171) (SEQ ID NO: 27) and Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); b) Histag-B3-nLuc(51-171)-mNeonGreen (SEQ ID NO: 28) and Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); c) i) His-B3-L (SEQ ID NO: 53) or ii) His-L-B3 (SEQ ID NO: 54) and i) Histag-B3-S-mNeonGreen (SEQ ID NO: 30) or ii) Histag-mNeonGreen-S- B3 (GSB3) (SEQ ID NO: 31); d) Histag-B3-L-mNeonGreen (SEQ ID NO: 32) and i) His-B3-S (SEQ ID NO: 51) or ii) His-S-B3 (SEQ ID NO: 52); e) His-L-I (SEQ ID NO: 56) and i) His-I-S-mNeonGreen (SEQ ID NO: 33), ii) His-I-mNeonGreen-S (SEQ ID NO: 34), or iii) His-Ihalf1-mNeonGreen-S- Ihalf2 (SEQ ID NO: 35). In some embodiments, at least one pair of fusion proteins comprises a pair of fusion proteins for Influenza. In some embodiments, the pair of fusion proteins for Influenza is selected from the following: i) His-84-L (SEQ ID NO: 74) or ii) L-84-His (SEQ ID NO: 75), and i) mCyRFP1-S-84-His (SEQ ID NO: 36), ii) S-mCyRFP1-84-His (SEQ ID NO: 37), iii) CyOFP1-S-84-His (SEQ ID NO: 38), iv) S-CyOFP1-84-His 6#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 (SEQ ID NO: 39), v) LSSmOrange-S-84-His (SEQ ID NO: 40), or v) S- LSSmOrange-84-His (SEQ ID NO: 41). In some embodiments, at least one pair of fusion proteins comprises a pair of fusion proteins for RSV. In some embodiments, the pair of fusion proteins for RSV is selected from the following: i) His-MED-L (SEQ ID NO: 49) or ii) His-L-MED (SEQ ID NO: 48), and i) MED-S-LSSmOrange-Histag (SEQ ID NO: 42), ii) Histag- LSSmOrange-S-MED (SEQ ID NO: 43), iii) MED-S-mCyRFP1-Histag (SEQ ID NO: 44), iv) Histag-mCyRFP1-S-MED (SEQ ID NO: 45), v) MED-S-CyOFP1-Histag (SEQ ID NO: 46), or v) Histag-CyOFP1-S-MED (RSMED) (SEQ ID NO: 47). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS FIG. 1 provides a schematic diagram showing the linear structure of construct Histag-B3-nLuc(51-171) (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 2 provides a schematic diagram showing the linear structure of construct Histag-B3-nLuc(51-171)-mNeonGreen (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 3 provides a schematic diagram showing the linear structure of construct Histag-B3-nLuc(6-50)-mNeonGreen (left panel) and the protein structure as predicted by AlphaFold (right panel). 7#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 FIG. 4 provides a schematic diagram showing the linear structure of construct Histag-B3-S-mNeonGreen (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 5 provides a schematic diagram showing the linear structure of construct Histag-mNeonGreen-S-B3 (GSB3) (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 6 provides a schematic diagram showing the linear structure of construct Histag-B3-L-mNeonGreen (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 7 provides a schematic diagram showing the linear structure of construct His-I-S-mNeonGreen (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 8 provides a schematic diagram showing the linear structure of construct His-I-mNeonGreen-S (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 9 provides a schematic diagram showing the linear structure of construct His-Ihalf1-mNeonGreen-S-Ihalf2(left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 10 provides a schematic diagram showing the linear structure of construct mCyRFP1-S-84-His (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 11 provides a schematic diagram showing the linear structure of construct CyOFP1-S-84-His (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 12 provides a schematic diagram showing the linear structure of construct S-CyOFP1-84-His (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 13 provides a schematic diagram showing the linear structure of construct LSSmOrange-S-84-His (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 14 provides a schematic diagram showing the linear structure of construct S-LSSmOrange-84-His (left panel) and the protein structure as predicted by AlphaFold (right panel). 8#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 FIG. 15 provides a schematic diagram showing the linear structure of construct MED-S-LSSmOrange-Histag (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 16 provides a schematic diagram showing the linear structure of construct Histag-LSSmOrange-S-MED (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 17 provides a schematic diagram showing the linear structure of construct MED-S-mCyRFP1-Histag (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 18 provides a schematic diagram showing the linear structure of construct Histag-mCyRFP1-S-MED (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 19 provides a schematic diagram showing the linear structure of construct MED-S-CyOFP1-Histag (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 20 provides a schematic diagram showing the linear structure of construct Histag-CyOFP1-S-MED (RSMED) (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 21 provides a schematic diagram showing the linear structure of construct His-MED-L (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 22 provides a schematic diagram showing the linear structure of construct His-L-MED (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 23 provides a schematic diagram showing the linear structure of construct His-S-MED (left panel) and the protein structure as predicted by AlphaFold (right panel). FIG. 24 is a schematic showing MiniRGB probes for same-well multicolor- based multiplexed detection of viruses with ultra-high bioluminescence resonance energy transfer (BRET) efficiency. Green fluorescent protein (GFP) and red fluorescent protein (RFP) are fused with the small luciferase fragment and binder targeting the trimeric assembly of the surface proteins from different viruses, respectively. 9#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 FIGS. 25A-25G. Engineered multicolor MiniRGB probes for the same-well multiplexed assay. FIG. 25A, Cartoon showing the multicolor-based multiplexed detection of influenza, COVID-19, and RSV via MiniRGB probes. FIG. 25B, Structure and Coomassie blue-stained SDS-PAGE showing MiniRGB probes fused with fluorescence proteins. Protein structures are predicted by AlphaFold. FIG. 25C, Emission spectra and images of MiniRGB probes for the multicolor-based multiplexed detection of SARS-CoV-2 spike proteins, influenza HA proteins, and RSV F proteins. FIG. 25D, BRET efficiency of MiniRGB probes with different designs for detecting SARS-CoV-2 spike proteins. FIG. 25E, Performance analysis of MiniRGB probes for the multicolor-based multiplexed detection of SARS-CoV-2 spike proteins and RSV F proteins. S / N: signal-to-noise ratio. FIGS. 25F And 25G, Same-well multicolor-based multiplexed detection of SARS-CoV-2 spike proteins and influenza HA proteins spiked in the clinical negative nasal wash. All the inserted images were taken by a cellphone (iPhone 13 Pro) camera. FIGS. 26A-26B. Bioluminescent probes for detecting RSV F proteins. FIG. 26A, Coomassie blue-stained SDS-PAGE showing the purity of SMED and LMED produced using mammalian cell expression system. Protein structures of SMED and LMED are predicted by AlphaFold. FIG. 26B, Binding analysis of SMED and LMED with RSV F proteins by microscale thermophoresis. FIGS. 27A-27H. Design of MiniRGB probes for multicolor-based multiplexed detection of viral surface proteins. FIGS. 27A-27D, Schematic representations illustrating the overlap of bioluminescence emission spectra with fluorescence protein (mNeonGreen, LSSmOrange, CyOFP1, and mCyRFP1) excitation and emission spectra. FIG. 27E, Proximity analysis of the two MiniRGB probe fragments bound to the viral surface proteins for detecting COVID-19, influenza, and RSV, respectively. The proximity is defined as 1 / D where D represents the distance between the C terminus of two B3 bound to SARS-CoV-2 spike proteins, N terminus of two SD84 (PDB ID: 6CNV) bound to influenza HA proteins16, and N terminus of two MED (PDB ID: 5UDC) bound to RSV F proteins15, respectively. FIG. 27F, The binding affinity of different MiniRGB probes to the viral surface proteins of COVID-19, influenza, and RSV, respectively. Affinity is defined as 1 / KDwhere KD represents the equilibrium dissociation constant. FIG. 27G, The overlap of bioluminescence emission spectra with fluorescence protein excitation spectra. FIG. 10#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 27H, The molecular brightness of the fluorescence proteins. The excitation and emission spectra, as well as the molecular brightness of fluorescence proteins are adopted from FPbase (www.fpbase.org / ). FIGS. 28A-I. SEC elution profiles of MiniRGB probes. The proteins were purified by size exclusion chromatography with Superdex 200 Increase 10 / 300 GL column. FIGS. 29A-29L. Performance of MiniRGB probes with different structure designs for multicolor-based multiplex detection of viral surface proteins. FIG. 29A, Summary of MiniRGB probes fused with green fluorescence proteins (mNeonGreen) for detecting SARS-CoV-2 spike proteins. FIG. 29B, Total bioluminescence intensity for detecting SARS-CoV-2 spike proteins using different pairs of MiniRGB probes. FIG. 29C, Emission spectra of different pairs of MiniRGB probes for detecting SARS-CoV-2 spike proteins. FIG. 29D, BRET efficiency of the MiniRGB probes with different structure designs for detecting spike proteins of SARS-CoV-2. FIG. 29E, Summary of MiniRGB probes fused with red fluorescence proteins (mCyRFP1), orange-red fluorescence proteins (CyOFP1), and orange fluorescence proteins (LSSmOrange) for detecting influenza HA proteins. FIG. 29F, Total bioluminescence intensity for detecting influenza HA proteins using different pairs of MiniRGB probes. FIG. 29G, Emission spectra of different pairs of MiniRGB probes for detecting influenza HA proteins. FIG. 29H, BRET efficiency of MiniRGB probes with different structure designs for detecting influenza HA proteins. FIG. 29I, Summary of MiniRGB probes fused with red fluorescence proteins (mCyRFP1), orange-red fluorescence proteins (CyOFP1), and orange fluorescence proteins (LSSmOrange) for detecting RSV F proteins. FIG. 29J, Total bioluminescence intensity for detecting RSV F proteins using different pairs of MiniRGB probes. FIG. 29K, Emission spectra of different pairs of MiniRGB probes for detecting RSV F proteins. FIG. 29L, BRET efficiency of MiniRGB probes with different structure designs for detecting RSV F proteins. All the inserted images were taken by a cellphone (iPhone 13 Pro) camera. The different BRET efficiency of MiniRGB probe pairs indicates the dependence of MiniRGB performance on the close proximity, binding affinity, spectra overlap, and molecular brightness, and order of protein domain. 11#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 FIG. 30. BRET efficiency of MiniRGB probes for detecting influenza HA proteins. mCyRFP1, CyRFP1, and LSSmOrange are fused with the small luciferase fragment and binders for detecting SARS-CoV-2 spike proteins with the emission of red light. S / N: sig. FIG. 31. A schematic showing that proximity is crucial for efficient bioluminescence generation at the concentration range of viral surface proteins in the patient’s body for clinical application. FIG. 32. Illustration of the Design of the HIV Luminescence Biosensor Detection System. Left panel is an illustration of the design for the HIV envelope glycoprotein trimer detection assay using the gp160 surface protein of HIV comprising a “double split” system employing the conformationally selective PCT145 antibody fragment (scFv) that binds to the quaternary epitope at the apex center of the native gp160 trimer of HIV. Eight constructs were engineered and purified that include a fragment of PGT145 in addition to a luciferase fragment. (Hence a “double split” system.) Right panel is a graphical representation of data showing that in the presence of the HIV trimer, these protein probes assemble an active luciferase and result in a 5-fold increase in the luminescence signal. DETAILED DESCRIPTION Bioluminescence resonance energy transfer (BRET) is useful in assays for studying protein-protein interactions via the energy transfer between a bioluminescent donor and an adjacent fluorescent acceptor30-32. Despite advances in developing various donor-acceptor pairs, most BRET sensors suffer from low BRET efficiency due to the existence of a high bioluminescence background, which might interfere with the signal readout and lead to low sensitivity. Here, we designed multicolor probes (the term probes is used interchangeably herein with “fusion proteins”) with ultra-high BRET efficiency for the multiplexed detection of analytes, e.g., multimeric viral surface proteins, by fusing fluorescence proteins with small luciferase fragments and corresponding binders targeting the trimeric assembly of viral surface proteins (FIG. 24). In the absence of viruses, split luciferase fragments of these probes remain inactive with low bioluminescence and fluorescence background. In the presence of viruses, the probes reconstitute functional luciferase enzyme at the apex of the 12#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 multimeric surface proteins, leading to the bioluminescent emission and the subsequent energy transfer to the adjacent fluorescent acceptors. The multicolor probes, one example of which is the MiniRGB probes described herein, possess ultra-high BRET efficiency based on several distinctive features. First, the high binding affinity between binders and viral surface proteins ensures a sufficient number of functional luciferase enzymes. Second, the trimeric assembly of viral surface proteins guarantees the close proximity (e.g., up to 1-2 nm, preferably less than 100 or less than 50 Angstrom) to reconstitute the functional luciferase enzyme with high enzymatic activity, thus generating bright blue bioluminescence to excite fluorescence acceptors. Third, the fusion of luciferase fragments with fluorescence proteins allows the close proximity of the donor-acceptor pair and the subsequent energy transfer from bioluminescent donors to fluorescent acceptors with high efficiency. Fourth, the fluorescence proteins with maximum overlap between the fluorescent excitation spectra and bioluminescent spectra, as well as high molecular brightness, are utilized to efficiently absorb the bioluminescent light and enhance the emitted light intensity from fluorescent acceptors, respectively. Fifth, flexible peptide linkers for both N- and C-terminus fusions facilitate the close proximity between donor and acceptor with minimized steric hindrance and provide opportunities to align their dipoles in an optimal orientation suitable for efficient energy transfer32. Additionally, small luciferase fragments with a low bioluminescence background are fused to fluorescence proteins, thus diminishing the excitation of the fluorescence background. Fusion Protein(s) Described herein are paired fusion proteins and their use for detecting and / or identifying one or more analytes, e.g., monomeric or multimeric protein analytes. Detection of more than one analyte is performed using a plurality of (i.e., more than one) pair of fusion proteins. For example, a sample can be contacted with more than one pair of fusion proteins to determine if the sample has one or more analytes present. Each pair of fusion proteins is specific for one analyte (although multiple pairs that are specific for the same analyte can also be used). For instance, to determine if the subject has SARS-CoV-2, Influenza, or RSV, a sample from a subject would be contacted with three pairs of fusion proteins. One pair is specific for 13#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 multimeric proteins expressed by SARS-CoV-2, one pair is specific for multimeric proteins expressed by Influenza, and one is specific for multimeric proteins expressed by RSV. Each pair of fusion proteins comprises i) a first fusion protein comprising one or more binding domains (which is specific for an analytes, e.g., a multimeric analyte, e.g., a multimeric analyte expressed by a pathogen, pathogenic organism, or cell, e.g., mammalian cell) fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding domains that bind to the same analyte as the first fusion protein fused to a second portion of the same reporter protein as the first fusion protein, wherein the first and second portions of the reporter protein, when together, comprise the reporter protein in its entirety and restores its function, wherein the first fusion protein and / or the second fusion protein further comprises a fluorophore (preferably, wherein only one of the fusion proteins comprises a the fluorophore). The components of the fusion proteins are described in detail as follows. First Fusion Proteins A first fusion protein(s) described herein comprises one or more binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), genetically fused to a first portion of a reporter protein as described herein. In some embodiments, the first fusion protein comprises, from C-terminal to N-terminal: (a) a first portion of a reporter protein, as described herein; and (b) one or more binding domains, as described herein. In some embodiments, the first or second fusion protein further comprises a fluorophore (and the other fusion protein optionally does not comprise a fluorophore). In those situations, the first fusion protein comprises, from C-terminal to N-terminal: (a) a first portion of a reporter protein, as described herein; (b) a fluorophore, and (c) one or more binding domains, as described herein. Alternatively, the first fusion protein comprises, from C-terminal to N-terminal: (a) a fluorophore, as described herein; (b) a first portion of a reporter protein, and (c) one or more binding domains, as described herein. In a further alternate embodiment, the first fusion protein comprises, from C-terminal to N- terminal: (a) one or more binding domains, as described herein, (b) a fluorophore, as described herein, and (c) a first portion of a reporter protein, as described herein. In a further alternate embodiment, the first fusion protein comprises, from C-terminal to 14#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 N-terminal: (a) one or more binding domains, as described herein, (b) a first portion of a reporter protein, as described herein, and (c) a fluorophore, as described herein. In a further alternate embodiment, the first fusion protein comprises, from C-terminal to N-terminal: (a) a first portion of a reporter protein, as described herein, (b) one or more binding domains, as described herein, and (c) a fluorophore, as described herein. In a further alternate embodiment, the first fusion protein comprises, from C-terminal to N-terminal: (a) a fluorophore, as described herein, (b) one or more binding domains, as described herein, and (c) a first portion of a reporter protein, as described herein. In some embodiments, the linker is from 1 to 50 amino acids long, e.g., in some embodiments the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long. In some embodiments, the linker comprises GSG, or variations thereof (e.g., SGGGGSGGGGSGGGGS (SEQ ID NO: 25)). Second Fusion Proteins A second fusion protein(s) described herein comprises one or more binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), genetically fused to a first portion of a reporter protein as described herein. In some embodiments, the first and second portions of the reporter protein, when together, comprise the reporter protein in its entirety (or at least comprises the function portion of the reporter protein in its entirety). In some embodiments, the second fusion protein comprises, from C-terminal to N-terminal: (a) a second portion of a reporter protein, as described herein; and (b) one or more binding domains, as described herein. In some embodiments, the second fusion protein further comprises a fluorophore (wherein, the first fusion protein optionally does not comprise a fluorophore). In those situations, the second fusion protein comprises, from C-terminal to N-terminal: (a) a second portion of a reporter protein, as described herein; (b) a fluorophore, and (c) one or more binding domains, as described herein. Alternatively, the second fusion protein comprises, from C- terminal to N-terminal: (a) a fluorophore, as described herein; (b) a second portion of a reporter protein, and (c) one or more binding domains, as described herein. In a 15#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 further alternate embodiment, the second fusion protein comprises, from C-terminal to N-terminal: (a) one or more binding domains, as described herein, (b) a fluorophore, as described herein, and (c) a second portion of a reporter protein, as described herein. In a further alternate embodiment, the second fusion protein comprises, from C- terminal to N-terminal: (a) one or more binding domains, as described herein, (b) a second portion of a reporter protein, as described herein, and (c) a fluorophore, as described herein. In a further alternate embodiment, the second fusion protein comprises, from C-terminal to N-terminal: (a) a second portion of a reporter protein, as described herein, (b) one or more binding domains, as described herein, and (c) a fluorophore, as described herein. In a further alternate embodiment, the second fusion protein comprises, from C-terminal to N-terminal: (a) a fluorophore, as described herein, (b) one or more binding domains, as described herein, and (c) a second portion of a reporter protein, as described herein. The second portion of the reporter protein of the second fusion protein is complementary to the first portion of the reporter protein of the first fusion protein, such that when the first and second fusion proteins are in proximity with one another the first and second portions of the reporter protein form a fully functional protein. In some embodiments, the first portion of the reporter protein is the smaller portion of the two (i.e., between the first and second portions of the reporter protein) and the second portion of the reporter is the larger portion of the two. In some embodiments, the first portion of the reporter protein is the larger portion of the two (i.e., between the first and second portions of the reporter protein) and the second portion of the reporter is the smaller portion of the two. “Close proximity,” as used herein, refers to a distance of up to 10 nm (e.g., up to 9 nm, up to 8 nm, up to 7 nm, up to 6 nm, up to 5 nm, up to 4 nm, up to 3 nm, up to 2 nm, or up to 1 nm), such as, up to 7-10 nm, up to 5-7 nm, up to 3-5 nm, up to 1-3 nm, up to 5-10 nm, up to 2-5 nm, or up to 1-2 nm, or a distance of less than 100 Å (e.g., less than 95 Å, less than 90 Å, less than 85 Å, less than 80 Å, less than 75 Å, less than 70 Å, less than 65 Å, less than 60 Å, less than 55 Å, less than 50 Å, less than 45 Å, less than 40 Å, less than 35 Å, less than 30 Å, less than 25 Å, less than 20 Å, less than 15 Å, less than 10 Å, less than 5 Å, or less than 1 Å), such as, about 95 Å to about 100 Å, about 90 Å to about 95 Å, about 85 Å to about 90 Å, about 80 Å to about 85 Å, about 75 Å to about 80 Å, about 70 Å to about 75 Å, about 65 Å to about 70 Å, about 60 Å to about 65 Å, about 55 Å to about 60 Å, about 50 Å to about 55 Å, 16#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 about 45 Å to about 50 Å, about 40 Å to about 45 Å, about 35 Å to about 40 Å, about 30 Å to about 35 Å, about 25 Å to about 30 Å, about 20 Å to about 25 Å, about 15 Å to about 20 Å, about 10 Å to about 15 Å, about 5 Å to about 10 Å, about 1 Å to about 5 Å (e.g., about 100 Å, about 95 Å, about 90 Å, about 85 Å, about 80 Å, about 75 Å, about 70 Å, about 65 Å, about 60 Å, about 55 Å, about 50 Å, about 45 Å, about 40 Å, about 35 Å, about 30 Å, about 25 Å, about 20 Å, about 15 Å, about 10 Å, about 5 Å, or about 1 Å). Thus, a first reporter protein fragment and a second reporter protein fragment, when in close proximity to each other (e.g., within a distance of up to 10 nm, ideally less than 50-60 Å from each other), can reconstitute a reporter protein in its entirety and restore its function (e.g., the reporter protein can generate a detectable signal in presence of a suitable substrate when the first and second reporter protein fragments come together). For example, a full and functional reporter protein can be formed when a first reporter protein fragment and a second reporter protein fragment come together, e.g., come within a distance of less than 10 nm, preferably within 50- 60 Å of each other. The importance of close proximity between the reporter protein fragments for generation of robust detectable signal by the reporter protein is also demonstrated, e.g., in FIG. 31. In some embodiments, the first fusion protein comprises a linker (e.g., a flexible linker) between the one or more binding domains, the first portion of the reporter protein, and, if present, the fluorophore. In some embodiments, the linker is from 1 to 50 amino acids long, e.g., in some embodiments the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long. In some embodiments, the linker comprises GSG, or variations thereof (e.g., SGGGGSGGGGSGGGGS (SEQ ID NO: 25)). Analytes Analytes detectable using the fusion proteins and methods described herein include proteins that assemble into multimeric complexes. Analytes can be expressed in pathogenic or non-pathogenic organisms. For example, analytes are one or more multimeric proteins expressed by mammalian cells, or pathogenic viruses, parasites, fungus, or bacteria. In some instances, the analytes, e.g., multimeric analytes, are expressed on the surface of a cell. In some instances, the analytes, e.g., multimeric 17#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 analytes, are expressed intracellularly (in these cases, the cell is lysed before being contacted / mixed / incubated). Although the present disclosure exemplifies multimeric proteins as analytes, monomeric proteins can also be detected using the present methods; the paired fusion proteins include binders that bind to adjacent but non- overlapping epitopes on the monomer, in close proximity to each other, sufficient to bring the complementary fragments of the reporter protein together. The analytes detectable using the fusion proteins and methods described herein can include multimeric proteins expressed by a pathogenic organism, preferably multimeric proteins that are present on the surface of the pathogenic organisms; multimeric proteins are those that assemble into multimeric complexes (e.g., dimers, trimers, tetramers, or pentamers). Therefore, pathogenic organisms include any virus, bacteria, parasites, archaea, protozoa or multicellular organism that utilize multimeric complexes on their surfaces. Viral surface proteins of enveloped viruses (e.g., SARS-CoV-2, RSV, Influenza, HIV, MERS-CoV, Ebola, Hepatitis C, MERS, Measles) assemble as homo-trimers, and such homo-trimeric viral proteins and, as such, can be detected by the fusion proteins of the present disclosure. Viral surface proteins (e.g., capsid proteins) of non-enveloped viruses (e.g., poliovirus, hepatitis E, BK virus, norovirus, rotavirus, coxsackievirus) also assemble as homo- pentamers, and such homo-pentameric viral proteins can also be detected by the fusion proteins of the present disclosure. In some embodiments, multimeric analytes that are detected by the present methods are one or more of multimeric eukaryotic membrane proteins. In some embodiments, multimeric analytes that are detected by the present methods are one or more of multimeric prokaryotic membrane proteins. Many bacterial surface proteins also assemble as multimers. Examples of useful multimeric proteins and their associated bacteria include, but are not limited to: 1) Type IV Pilus (T4P) Proteins: Many bacteria, including Pseudomonas aeruginosa and Neisseria gonorrhoeae, produce Type IV pili, which are important for adherence and motility. 2) Pilin (Fimbrial Protein) or Porin Proteins (PorB): important in Neisseria gonorrhoeae. 3) CyaA (Adenylate Cyclase) Protein: This protein from Bordetella pertussis is involved in the pathogenic mechanisms of the bacterium. CyaA can form a trimeric structure, which is essential for its function as it interacts with host cells. 4) Aerobactin Irom Acquisition System: In Escherichia coli, the 18#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 component proteins that are part of the aerobactin system, which plays a role in iron acquisition, may assemble in trimers to facilitate transport and binding. 5) Collagen- Binding Protein (CbpA): Found in Streptococcus pneumoniae, this surface protein that binds to collagen can also exist in a trimeric form, which is crucial for its function in host tissue adhesion. 6) Mannose-Binding Pili: In some strains of E. coli, proteins that form the mannose-binding pili can organize into trimers to enhance the linking efficiency to host tissues. 7) Chlamydial major outer membrane protein (MOMP): in Chlamydia trachomatis MOMP forms homo^trimers (see, Feher VA, Randall A, Baldi P, Bush RM, de la Maza LM, Amaro RE (2013) A 3-Dimensional Trimeric ^-Barrel Model for Chlamydia MOMP Contains Conserved and Novel Elements of Gram- Negative Bacterial Porins. PLoS ONE 8(7): e68934. https: / / doi.org / 10.1371 / journal.pone.0068934 and Peterson EM, Cheng X, Markoff BA, Fielder TJ, de la Maza LM. Functional and structural mapping of Chlamydia trachomatis species-specific major outer membrane protein epitopes by use of neutralizing monoclonal antibodies. Infect Immun. 1991 Nov;59(11):4147-53; each incorporated herein by reference in their entireties). 8) TprK (Treponema pallidum repeat protein K): in Treponema pallidum. 9) NaP, MgPa, ML1 and ML2: in Mycoplasma genitalium. Thus, in some embodiments, multimeric analytes that are detected by the present methods are one or more of multimeric bacterial proteins. Parasitic surface proteins can also assemble into multimeric complexes. An exemplary parasite includes malaria (merozoite surface proteins, MSP-1 and MSP-2). Another example is Trichomonas vaginalis (Target: P270 or flagellar proteins). Thus, in some embodiments, multimeric analytes that are detected by the present methods are one or more of multimeric fungal proteins. Examples of binding domains for analytes that can be used with the methods of the disclosure include, but are not limited to, the following. Exemplary Binding Domains Analytes that can be detected by the fusion proteins of the present disclosure can include multimeric analytes (e.g., multimeric proteins). In certain embodiments, binding domains of the fusion proteins mimic or comprise domains from binders that recognize non-overlapping epitopes at or near the apex of a multimeric analyte. Additionally, or in the alternative, binding domains of the fusion proteins can be 19#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 binders that recognize non-overlapping epitopes at or near the apex of a multimeric analyte. Additionally, or in the alternative, binding domains of the fusion proteins can be fragments of binders that recognize non-overlapping epitopes at or near the apex of a multimeric analyte. It should be appreciated that the following binding domains exemplify embodiments of the methods disclosed herein. It should be appreciated that other binding domains that could be designed and used with the methods of the present disclosure are also contemplated for use herein. Additional exemplary binding domains can be found in International Publication No. WO2025 / 064581 A1, which is incorporated by reference herein in its entirety. Binding Domains for SARS-CoV-2 Binding domains of these fusion proteins (e.g., fusion proteins that can be used for detection of SARS-CoV-2 by the present methods) can mimic or comprise angiotensin-converting enzyme 2 (ACE2) receptors on host cells that bind to the receptor binding domain (RBD) of SARS-CoV-2 spike (e.g., SARS-CoV-2 wild-type spike). Additionally, or in the alternative, these binding domains can be an ACE2 receptor (from host cells) or fragments thereof that binds to the RBD of SARS-CoV-2 spike (e.g., SARS-CoV-2 wild-type spike). In some embodiments, binding domains of these fusion proteins can be binders (or fragments thereof) that recognize and bind to epitopes (e.g., non-overlapping epitopes) at or near the apex of the trimeric assembly of the SARS-CoV-2 spike protein (e.g., SARS-CoV-2 wild-type spike). These fusion proteins (e.g., fusion proteins for detection of SARS-CoV-2 by the present methods) can comprise one or more of the following binding domains: B3, I, Ihalf1, Ihalf2, CTC, CT30, ACE2. B3, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 10; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 10 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 10. I, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 20#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 11; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 11 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 11. Ihalf1, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 12; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 12 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 12. Ihalf2, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 13; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 13 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 13. CTC, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 14; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 14 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 14; CT30, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 15; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 15 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 15; and 21#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 ACE2, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 16 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 16. Binding domains for Influenza Binding domains of these fusion proteins (e.g., fusion proteins that can be used for detection of influenza by the present methods) can mimic or comprise domains of multiple-domain binders that target multiple highly conserved epitopes of trimeric hemagglutinin (HA) proteins of influenza A and B viruses. Additionally, or in the alternative, these binding domains can be multiple-domain binders that target multiple highly conserved epitopes of trimeric HA proteins of influenza A and B viruses. In some embodiments, the multiple-domain binders comprise binders that target influenza A and binders that target influenza B. For example, a fusion protein can comprise one or more binding domains that mimic or comprise domains of multiple-domain binders, wherein the multiple-domain binders comprise two binders (e.g., SD36 and SD38) that target influenza A and two binders (e.g., SD83 and SD84) that target influenza B. Additionally, or in the alternative, a fusion protein can comprise one or more binding domains that are multiple-domain binders or fragments thereof, wherein the multiple-domain binders comprise two binders (e.g., SD36 and SD38) that target influenza A and two binders (e.g., SD83 and SD84) that target influenza B. One or more of these binders can recognize the HA stem and / or the HA head. For example, each of SD36, SD38, and SD83 recognizes the HA stem, and SD84 binds to a conserved epitope in the HA head. In some embodiments, binding domains of these fusion proteins can be binders (or fragments thereof) that recognize and bind to epitopes (e.g., non-overlapping epitopes) at or near the apex of the trimeric assembly of the influenza HA protein. These fusion proteins (e.g., fusion proteins for detection of influenza virus by the present methods) can comprise one or more of the following binding domains: SD84, SD83, SD36, SD38. 22#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 SD84, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 17; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 17 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 17. In some embodiments, SD84 targets influenza B. In some embodiments, SD84 binds to a conserved epitope in the HA head (e.g., the HA head of influenza B). SD83, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 18; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 18 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 18. In some embodiments, SD83 targets influenza B. In some embodiments, SD83 recognizes the HA stem (e.g., the HA stem of influenza B). SD36, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 19; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 19 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 19. In some embodiments, SD36 targets influenza A. In some embodiments, SD36 recognizes the HA stem (e.g., the HA stem of influenza A). SD38, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 20; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 20 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 20. In some embodiments, SD38 targets influenza A. In some embodiments, SD38 recognizes the HA stem (e.g., the HA stem of influenza A). 23#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Binding domains for RSV Binding domains of these fusion proteins (e.g., fusion proteins that can be used for detection of RSV by the present methods) can recognize and bind to the trimeric fusion glycoprotein (F), a surface protein of RSV. In some embodiments, these binding domains recognize and bind to the apex of the trimeric fusion glycoprotein (F) with high affinity. In some embodiments, binding domains of these fusion proteins are binders (or fragments thereof) that recognize and bind to epitopes (e.g., non-overlapping epitopes) at or near the apex of the trimeric assembly of the RSV fusion glycoprotein (F). These fusion proteins (e.g., fusion proteins for detection of RSV by the present methods) can comprise the following binding domain: MED97 (also referred to herein as “MED” or “M97”), a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 21 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 21. Binding domains for HIV HIV-1 envelope glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. Binding domains of these fusion proteins (e.g., fusion proteins that can be used for detection of HIV by the present methods) can mimic, or can be, one or more antibody fragments, e.g., single-chain variable fragments (scFvs), that bind to HIV envelope glycoprotein gp160. For example, binding domains of these fusion proteins can mimic, or can be, one or more antibody fragments (e.g., scFvs) that bind to quaternary epitopes at the apex center of the native gp160 trimer of HIV. Thus, in some embodiments, binding domains of these fusion proteins can comprise one or more fragments (e.g., scFvs) of PGT145. A PGT145 fragment (e.g., PGT145 scFv) for use in the present methods can be a variable light fragment of PGT145 (also referred to herein as PGT145 (light or variable light chain)) and / or a heavy fragment of PGT145 (also referred to herein as PGT145 (heavy or variable heavy chain)). Preferably, the binding domain for the PGT145 is split across a pair of fusion 24#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 proteins, such that the variable heavy chain is the binding domain on a first fusion protein, and the variable light chain is the binding domain on a second fusion protein (or vice versa). Such double-split fusion proteins in which both the reporter protein and the first binding domain are split are used in pairs such that the binding of the first and second fusion protein results in reconstitution of the binding domain (forming scFv) and of the reporter protein. A schematic representation of fusion proteins comprising the double split is provided in FIG. 32. Additionally, fusion proteins for HIV can further binding domains that can mimic, or can be, one or more fragments of antibodies (e.g., BN117, 10E8), and / or, one or more domains from CD4 receptor, which is the host receptor that binds to HIV envelope glycoprotein gp120. These fusion proteins (e.g., fusion proteins for detection of HIV by the present methods) can comprise one or more of the following binding domains: PGT145 (variable light chain), a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 76; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 76 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 76; PGT145 (variable heavy chain), a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 77; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 77 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 77; BN117 scFv, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 78; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 78 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 25#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 78; 10E8 scFv, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 79; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 79 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 79; and CD4 receptor, a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 80; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 80 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 80. Binding domains for Poliovirus Binding domains of these fusion proteins (e.g., fusion proteins that can be used for detection of poliovirus by the present methods) can mimic, or can be, fragments of one or more binders of poliovirus. These binders can recognize and bind to viral capsid proteins (e.g., VP1, VP3, VP2, and / or VP4). The poliovirus capsid contains 60 copies each of the four viral polypeptides VP1, VP2, VP3, and VP4. The arrangement of proteins in the capsid creates poliovirus’s icosahedral symmetry. The virion surface is covered with star-shaped mesas at its five-fold axes surrounded by deep canyons and three-bladed propellers. These are situated at three-fold axes separated by saddle depressions straddling two-fold axes. Capsid proteins VP1, VP2, and VP3 all have an eight-stranded ^-barrel fold but have different shaped loops on their N- and C-terminal extensions. In some embodiments, the binder of poliovirus comprises CD155 (also known as poliovirus receptor (PVR)), which is the receptor of poliovirus on host cells. Thus, in some embodiments, binding domains of these fusion proteins can comprise one or more fragments of CD155 (e.g., one or more fragments of CD155 ectodomain). In 26#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 some embodiments, the binder of poliovirus comprises one or more antibodies (e.g., A12 and F12) that can neutralize polioviruses from both serotypes 1 and 2. Cryo-EM studies shows that five Fab of the A12 antibody can bind simultaneously at the sites located at the bottom of the canyon surrounding the fivefold axis of symmetry. Thus, in some embodiments, binding domains of these fusion proteins comprise one or more fragments of A12 and / or F12. These fusion proteins (e.g., fusion proteins for detection of poliovirus by the present methods) can comprise one or more of the following binding domains: CD155 fragment (also referred to herein as CD155 domain or CD155), a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 91; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 91 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 91; A12 fragment (also referred to herein as A12 domain), a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 96; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 96 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 96; and F12 fragment (also referred to herein as F12 domain), a binding domain that comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 101; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 101 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids; and / or the amino acid sequence of SEQ ID NO: 101. Binding Domain for Chlamydia trachomatis Binding domains of these fusion proteins (e.g., fusion proteins that can be used for detection of Chlamydia trachomatis by the present methods) can recognize 27#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 and bind to the Major outer membrane porin, serovar C (MOMP), a surface protein of Chlamydia trachomatis. MOMP comprises about 60% of the outer membrane protein mass of Chlamydia Trachomatis. In some embodiments, the very high copy number of MOMP on the surface of the bacteria results in a strong antigen signal using the present detection methods even if only a small number of bacteria are present in the sample from a subject. In some embodiments, the amino acid sequence of MOMP is SEQ ID NO: 125 (Uniprot Accession No. P08780): MKKLLKSVLVFAALSSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPCTT WCDAISMRVGYYGDFVFDRVLKTDVNKEFQMGAAPTTSDVAGLQNDPTINV ARPNPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATTGYLKGNSAS FNLVGLFGTKTQSSSFNTAKLIPNTALNEAVVELYINTTFAWSVGARAALWE CGCATLGASFQYAQSKPKVEELNVLCNASEFTINKPKGYVGAEFPLNITAGTE AATGTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRVSFDADTIRIAQPK LAEAILDVTTLNRTTAGKGSVVSAGTDNELADTMQIVSLQLNKMKSRKSCGI AVGTTIVDADKYAVTVEARLIDERAAHVNAQFRF (SEQ ID NO: 125). In some embodiments, a binding domain recognizes and binds to the surface- exposed loops at the apex of the MOMP with high affinity. In some embodiments, a binding domain of these fusion proteins are binders (or fragments thereof) that recognize and bind to epitopes (e.g., non-overlapping epitopes) at or near the surface- exposed loops at the apex of the Chlamydia Trachomatis MOMP. In some embodiments, fusion proteins (e.g., fusion proteins for detection of Chlamydia Trachomatis by the present methods) can comprise the following binding domain: EVLPEEDEDEE (SEQ ID NO: 126). In some embodiments, a MOMP binding domain comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 126; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 126 by 1, 2, 3, 4, or 5 amino acids; and / or the amino acid sequence of SEQ ID NO: 126. Binding Domain for Neisseria meningitidis Binding domains of these fusion proteins (e.g., fusion proteins that can be used for detection of Neisseria meningitidis by the present methods) can recognize 28#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 and bind to the PorB, a major outer membrane porin protein of Neisseria meningitidis. PorB forms a trimeric structure with a pore that facilitates the diffusion of small, essential molecules into the cell. In some embodiments, the amino acid sequence of PorB is SEQ ID NO: 127 (PMID: 23255122): MASMTGGQQMGRDLQVTLYGTIKAGVEVSRVKDAGTYKAQGGKSKTATQI ADFGSKIGFKGQEDLGNGMKAIWQLEQKASIAGTNSGWGNRQSFIGLKGGFG TVRAGNLNTVLKDSGDNVNAWESGSNTEDVLGLGTIGRVESREISVRYDSPV FAGFSGSVQYVPRDNANDVDKYKHTKSSRESYHAGLKYENAGFFGQYAGSF AKYADLNTDAERVAVNTANAHPVKDYQVHRVVAGYDANDLYVSVAGQYE AAKNNEVGSIKGKKHEQTQVAATAAYRFGNVTPRVSYAHGFKAKVNGVKD ANYQYDQVIVGADYDFSKRTSALVSAGWLKQGKGAGKVEQTASMVGLRHK F (SEQ ID NO: 127). In some embodiments, a binding domain recognizes and binds to the surface- exposed loops at the to the apex of the PorB with high affinity. In some embodiments, a binding domain of these fusion proteins are binders (or fragments thereof) that recognize and bind to epitopes (e.g., non-overlapping epitopes) at or near the surface- exposed loops at the apex of the Neisseria meningitidis PorB. In some embodiments, fusion proteins (e.g., fusion proteins for detection of Neisseria meningitidis by the present methods) can comprise the following binding domain: GAAVAAENAAKAAAKKAAADAAAKAANA (SEQ ID NO: 128). In some embodiments, a PorB binding domain comprises: an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 128; an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 128 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids; and / or the amino acid sequence of SEQ ID NO: 128. Reporter Protein In some cases, the systems and methods described herein utilize reporter protein. As noted above, each fusion protein comprises a portion (i.e., fragment) of a reporter protein, such that the first and second fusion proteins comprise complementary fragments to one another. Accordingly, when a first fusion protein is 29#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 in proximity with a complementary second fusion protein, the pair forms a complete (or full length) and functional reporter protein. Thus, for use in the present methods, a reporter protein is split into two fragments, e.g., two complementary fragments. These complementary fragments can be referred to herein as reporter protein fragments, split reporter protein fragments, first and second reporter protein fragments, and / or first and second split reporter protein fragments. The reporter protein fragments are complementary to each other, e.g., the first reporter protein fragment is complementary to the second reporter protein fragment and vice versa. A full and functional reporter protein is formed when the two complementary reporter protein fragments come in close proximity of one another (e.g., when the binding domains of the first and second fusion proteins bind to their corresponding target analyte in a sample). In some embodiments, the reporter protein is an enzyme, e.g., a protein that can generate detectable signals through enzymatic reactions in an assay. In some embodiments, the reporter protein (e.g., enzyme) is luminescent, e.g., can produce light upon interaction with a luminogenic substrate. Preferably, luciferase is used; alternative enzymes that can be used as reporter proteins include, without limitation: beta-lactamase, beta-galactosidase, beta-glucuronidase, horseradish peroxidase (HRP), chloramphenicol acetyl transferase (CAT), and alkaline phosphatase. In some embodiments, luciferase is used as a reporter protein in the present methods. Bioluminescence is a natural phenomenon of living organisms creating their own light. The basis of bioluminescence is the interaction of the enzyme luciferase with a luminogenic substrate (e.g., luciferin) to produce light. The luciferases that are used most widely are beetle luciferases (including firefly luciferase), Renilla luciferase, and a modified deep sea shrimp luciferase (NanoLuc® luciferase). Luciferase genes have been cloned from bacteria, beetles (e.g., firefly), Renilla, deep sea shrimp (Oplophorus), Aequorea, Vargula and Gonyaulax (a dinoflagellate). Of these, only luciferases from bacteria, beetles, deep sea shrimp and Renilla have found general use as reporter genes to assess transcriptional expression. Thus, in some embodiments, the present methods use NanoLuc® luciferase as a reporter protein. Luciferase (e.g., NanoLuc®) can react with engineered luciferin (fluorofurimazine, FFz) with increased aqueous solubility and 30#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 generate bright blue bioluminescence with ultrahigh sensitivity and high stability for detection of target analytes in samples. In some embodiments, luciferase (e.g., NanoLuc®) is split into two complementary fragments. These fragments can be referred to herein as split luciferase fragments. In some embodiments, one of the two split luciferase fragments is a large fragment, and the other fragment is a small fragment. In some embodiments, the large fragment (also referred to herein as the large luciferase fragment and / or the large split luciferase fragment) comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the large luciferase fragment comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the large luciferase fragment comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the large luciferase fragment comprises 158 amino acids. Such large luciferase fragments can be referred to herein as split luciferase 158 amino acid fragment, luciferase 158 amino acid fragment, and / or split luciferase fragment 158 amino acid. In some embodiments, the small fragment (also referred to herein as the small luciferase fragment and / or the small split luciferase fragment) comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the small luciferase fragment comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 by 1, 2, 3, 4, or 5 amino acids. In certain embodiments, the small luciferase fragment comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the small luciferase fragment comprises 11 amino acids. Such small luciferase fragments can be referred to herein as split luciferase 11 amino acid fragment, luciferase 11 amino acid fragment, and / or split luciferase fragment 11 amino acid. In some embodiments, the first fusion protein comprises the large luciferase fragment and the second fusion protein comprises the small luciferase pair. In some 31#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 embodiments, the first fusion protein comprises the small luciferase fragment and the second fusion protein comprises the large luciferase pair. In some embodiments, luciferase (e.g., NanoLuc®) is split into two complementary fragments. These fragments can be referred to herein as split luciferase fragments. In some embodiments, one of the two split luciferase fragments is a large fragment (referred to as nLuc(51-171)), and the other fragment is a small fragment (referred to as nLuc(6-50)). In some embodiments, the nLuc(51-171) comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nLuc(51- 171) fragment comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the nLuc(51- 171) fragment comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nLuc(51-171) fragment comprises 120 amino acids. In some embodiments, the nLuc(6-50) comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the nLuc(6-50) fragment comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 4 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the nLuc(6-50) fragment comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the nLuc(6-50) fragment comprises 46 amino acids. Additionally, or in the alternative, fusion proteins of the present disclosure can comprise beta-lactamase as reporter protein. The activity of beta-lactamase, a bacterial enzyme, is detected using colorimetric or fluorescence substrates. To produce split enzyme, the protein is cut between Gly196 and Leu198 resulting in two inactive fragments. See, e.g., Galarneau et al. (Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein interactions. Nat Biotechnol. 2002, 20(6):619-22). 32#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Additionally, or in the alternative, fusion proteins of the present disclosure can comprise beta-galactosidase as reporter protein. ^-galactosidase converts lactose to galactose and glucose and has been used as a reporter in several molecular biology applications. See, e.g., Broome et al. (Expanding the utility of beta- galactosidase complementation: piece by piece. Mol Pharm. 2010;7(1):60-74). When lactose in replaced with a colorless substrate like X-Gal, this enzyme converts it to a blue compound. Fluorescent and chemiluminescent substrates are also available for this enzyme. ^-galactosidase was split into a small N terminal fragment (^-donor) and the large remaining subunit (^-acceptor). The ^-acceptor complements into an active enzyme only when ^-donor is present. Additional enzymes that can be used as reporter proteins in the fusion proteins of the present method include, without limitation, beta-glucuronidase (see, Geddie and Matsumura, Antibody-induced oligomerization and activation of an engineered reporter enzyme. J Mol Biol. 2007, 369(4):1052-9); Dihydrofolate reductase (see, Remy et al. Detection of protein-protein interactions using a simple survival protein- fragment complementation assay based on the enzyme dihydrofolate reductase, Nat Protoc. 2007, 2(9):2120-5); Protein Phosphatase 1 (see, De Munter et al. Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett. 2017, 591(2):415-24); Biotin ligase (see, Bertolotti, The split protein phosphatase system. Biochem J. 2018, 475(23):3707-23); and Split SNAPS-tag (see, Mie et al., Tracking a protein following dissociation from a protein-protein complex using a split SNAP-tag system. Anal Biochem. 2015, 477:53-5). Fluorophores As noted above, in each pair of fusion proteins, one (either the first or the second fusion protein) further comprises a fluorophore (also referred to herein as “fluorescent acceptor domain” or “acceptor molecule”). The fluorophore, as contemplated herein, refers to a compound which can accept energy emitted as a result of the activity of a chemiluminescent donor (e.g., the reporter protein, such as luciferase), and re-emit it as light energy. Acceptor molecules are well known in the art, especially those suitable for Bioluminescence resonance energy transfer (BRET). Reporter protein / fluorophore pairs are chosen such that the emission spectra of the reporter protein overlap with the excitation spectra of the fluorophore, such that light 33#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 emitted by the reporter is absorbed by the fluorophore, resulting in fluorescence from the fluorophore. Additionally, the fluorophore is attached to fusion protein that has the smaller portion of a reporter protein (e.g., the smaller luciferase fragment). In some instances, the fluorophore is attached to both fusion proteins of each pair of fusion proteins. A fluorophore and a reporter protein are selected that exhibit sufficient overlap of the emission spectrum (e.g., of the reporter protein) and the excitation spectrum (e.g., of the fluorophore) to provide efficient energy transfer between the two (e.g., by nonradiative dipole-dipole coupling). In some embodiments, the peak emission of the reporter protein is substantially separated in wavelength from the peak emission of the fluorophore, e.g., by at least 80nm, 100nm, 120nm, 140nm, etc. The acceptor molecules can be a protein. Examples include, but are not limited to, green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Venus, mOrange, Topaz, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, t-dimer2, t- dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein or a Phycobiliprotein, or a biologically active variant or fragment of any one thereof. In some embodiments, the fluorophore is mNeonGreen and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the mNeonGreen comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 5 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the mNeonGreen comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the fluorophore is mCyRFP1 and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the mCyRFP1 comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 6 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 34#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the mCyRFP1 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fluorophore is CyOFP1 and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the CyOFP1 comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 7 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the CyOFP1 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the fluorophore is LSSmOrange and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the LSSmOrange comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 8 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the LSSmOrange comprises the amino acid sequence of SEQ ID NO: 8. The acceptor molecules can be non-proteinaceous. In some instances, the fluorophore is a small molecule. Examples of acceptor molecules that are not proteins include, but are not limited to, Alexa Fluor dye, Bodipy dye, Cy dye, fluorescein, dansyl, umbelliferone, fluorescent microsphere, luminescent microsphere, fluorescent nanocrystal, Marina Blue, Cascade Blue, Cascade Yellow, Pacific Blue, Oregon Green, Tetramethylrhodamine, Rhodamine, Texas Red, rare earth element chelates, or any combination or derivatives thereof. The fusion proteins can include one or more than one (of the same type) small molecule fluorophore. In some instances, the reporter protein is luciferase, and the fluorophore is one of the following: mNeonGreen, mCyRFP1, CyOFP1, or LSSmOrange. In some instances, a fusion protein comprises the smaller luciferase fragment and one of the following: mNeonGreen, mCyRFP1, CyOFP1, or LSSmOrange. In some instances, the portion of the reporter protein and the fluorophore are separated by a short linker (e.g., 1 to 20 amino acids long, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 35#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 17, 18, 19, or 20 amino acids long. In some embodiments, the linker comprises GSG, or variations thereof (e.g., SGGGGSGGGGSGGGGS (SEQ ID NO: 25)). Purification Tags The recombinant or synthetic polypeptides of the invention can also comprise purification moieties or tags to facilitate their purification (e.g., prior to use in the methods and uses of the invention). Any suitable purification moiety or tag can be incorporated into the polypeptide and such tags / moieties are well known in the art. For instance, in some embodiments, the recombinant or synthetic peptide can comprise a peptide purification tag or moiety, e.g., a His-tag sequence. The purification tags can be incorporated at any position within the fusion proteins. In some embodiments, the purification moiety is located at or towards (i.e., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of) the N- or C-terminus of the polypeptide. For example, fusion proteins of the present disclosure can comprise a purification tag at the N-terminal. Additionally, or in the alternative, fusion proteins of the present disclosure can comprise a purification tag at the C-terminal. A cleavable linker can be used to attach the purification tag if removal is desired. In some embodiments, the purification tag is a histidine tag (e.g., a 6X- histidine tag) and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the purification tag is a FLAG, a c-Myc tag, or a biotin tag. Linkers In some embodiments, the fusion proteins of the present disclosure comprise one or more linkers. For example, each component (e.g., the binding domain, the portion of the reporter protein, the fluorophore, the purification tag) of the fusion proteins can be fused to one another via a linker. Additionally, or in the alternative, linkers may be present between two binding domains. In some embodiments, the linker is from 1 to 50 amino acids long, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 36#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long. In some embodiments, the linker comprises GSG, or variations thereof (e.g., SGGGGSGGGGSGGGGS (SEQ ID NO: 25)). Linkers are described, for example, in Argos, Mol Biol 211:943–958, 1990; George and Heringa, Protein Eng 15:871–879, 2002; Chen et al., Biotechniques 49:513–518, 2010; and Chen et al., Adv Drug Deliv Rev 65(10):1357-1369, 2013. A linker for use in a fusion protein described herein can be a flexible linker, a rigid linker, and / or an in vivo cleavable linker. For example, a linker for use in a fusion protein described herein can be a flexible linker. Flexible linkers are usually applied when the protein domains that need to be joined require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce the unfavorable interaction between the linker and the protein moieties. An example of the most widely used flexible linker is the sequence (GGGGS)n (SEQ ID NO: 22). Additionally, or in the alternative, a linker for use in a fusion protein described herein can be a rigid linker. While flexible linkers have the advantage of connecting the functional domains passively and permitting a certain degree of movement, the lack of rigidity of these linkers can be a limitation. There are several examples in the literature where the use of flexible linkers resulted in poor expression yields or loss of biological activity. Under such situations, rigid linkers can be successfully applied to keep a fixed distance between the domains and to maintain their independent functions. Rigid linkers exhibit relatively stiff structures by adopting ^-helical conformations or by containing multiple Pro residues. Examples of some rigid linkers are: (EAAAK)n (SEQ ID NO: 23) and (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu. Additionally, or in the alternative, a linker for use in a fusion protein described herein can be an in vivo cleavable linker. Flexible and rigid linkers represent stable linkers that covalently join functional protein domains together to act as one molecule 37#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 throughout the in vivo processes that the component protein(s) are involved in. This stable linkage between functional domains provides many advantages such as a prolonged plasma half-life (e.g., albumin or Fc-fusions). However, it also has several potential drawbacks, including, steric hindrance between functional domains, decreased bioactivity, and altered biodistribution and metabolism of the protein moieties due to the interference between domains. Under such circumstances, cleavable linkers are used to release free functional domains in vivo. This type of linker may reduce steric hindrance, improve bioactivity, or achieve independent actions / metabolism of individual domains of recombinant fusion proteins after linker cleavage. The design of in vivo cleavable linkers in recombinant fusion proteins is quite challenging. Unlike the versatility of crosslinking agents available for chemical conjugation methods, linkers in recombinant fusion proteins must necessarily be oligopeptides. For example, an in vivo cleavable disulfide linker (LEAGCKNFFPR^SFTSCGSLE) (SEQ ID NO: 24) (the arrow indicates where cleavage occurs), based on the reversible nature of the disulfide bond, was designed for recombinant fusion proteins by Chen et al. (Biotechniques 49:513–518, 2010), and offered the advantage of generating a precisely constructed, homogeneous product by recombinant methods. All of the fusion proteins described herein can be generated using standard molecular biological procedures, e.g., for manipulating and expressing recombinant DNA. See, e.g., Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) John Wiley & Sons (1995), and Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (June 15, 2012) and supplements thereof, and other standard laboratory manuals. The fusion proteins can be expressed, e.g., stably expressed, in a bacterial expression system (e.g., E. coli) and / or a mammalian expression system (e.g., in HEK293 cells). Table 1: Sequences of Exemplary Components of the Fusion Proteins Described above 38 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 39 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 40 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 41 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Table 2: Sequences of Exemplary Fusion Proteins 42 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 43 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 44 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 45 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 46 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 47 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 48 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 49 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 50 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 51 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 52 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 53 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 54 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 55 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Compositions Comprising Fusion Proteins Also provided herein are compositions comprising one or more fusion proteins of the present disclosure. In some embodiments, the composition is a liquid composition. For example, liquid compositions comprising one or more fusion proteins (e.g., a pair of fusion proteins, such as a first fusion protein and a second fusion protein) can be used for detecting analytes by the present methods. In some embodiments, the composition is lyophilized. For example, a composition (e.g., liquid composition) comprising one or more fusion proteins (e.g., a pair of fusion proteins, such as a first fusion protein and a second fusion protein) can be lyophilized for use in a system (e.g., a kit for detection of analytes by the present methods). In some embodiments, the composition can comprise more than one pair of fusion proteins. For example, the composition can comprise a combination of the following: a pair of fusion proteins for SARS-CoV-2, a pair of fusion proteins for influenza virus, and / or a pair of fusion proteins for RSV. The concentration of each fusion protein (e.g., each of the first and second fusion proteins) in the composition can be in a range from 0.01 nM to 1 M (e.g., 0.01 nM, 0.05 nM, 0.10 nM, 0.15 nM, 0.20 nM, 0.25 nM, 0.30 nM, 0.35 nM, 0.40 nM, 0.45 nM, 0.50 nM, 0.55 nM, 0.60 nM, 0.65 nM, 0.70 nM, 0.75 nM, 0.80 nM, 0.85 nM, 0.90 nM, 0.95 nM, 1.0 M, or any increment in between). The composition can comprise a mixture of buffers, solvents, and / or excipients necessary for maintaining the stability of the fusion protein. Methods for Detecting and Identifying One or More Analytes The fusion proteins of the present disclosure can be used for detection of one or more analytes, e.g., multimeric analytes, such as proteins expressed by pathogens, pathogenic organisms, or cells (e.g., mammalian cells). Multimeric protein analytes refer to proteins that require assembly of more than one individual protein subunits 56 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 into multimeric complexes. In some embodiments, analytes that are detected by the present methods are one or more of multimeric surface proteins. Additionally, as noted above, the fusion proteins described herein allow for the multiplex detection of organisms (or multimeric analytes) by fusing fluorescence proteins with small luciferase fragments and corresponding binders (i.e., binding domains) targeting the analytes, e.g., multimeric assembly (e.g., of pathogenic organism surface proteins). In the absence of the analyte, split luciferase fragments of the fusion proteins are inactive with low bioluminescence and fluorescence background. In the presence of the analytes, the fusion proteins reconstitute functional luciferase enzyme at the apex of the multimeric (e.g., more than one) surface proteins, leading to the bioluminescent emission and the subsequent energy transfer to the adjacent fluorescent acceptors. Depending on the fluorescent acceptor, the visualized colorimetric readout can detect and identify which analyte (and thus which pathogenic organism, where the analytes are associated with pathogenic organisms) is present. Use of different fluorescent acceptors with distinguishable (i.e., non-overlapping) emissions spectra for each analyte not only allows the detection and identification of a single analyte but also the detection and identification of multiple analytes, should more than one analyte be present in a sample. Accordingly, provided herein are methods for detecting and identifying one or more analytes in a sample. “Samples” (or “sample” or “test sample”) refers to biological and non-biological samples. Samples can include any environmentally obtained sample, e.g., air, waste-water, drinking water, animal watering or feed troughs, marine water, lake water, pond water, puddles, tanks, or water supply reservoir. Other examples include swabs from surfaces (e.g., cooking and / or dining surface), food samples, cosmetic products, pharmaceutical products, fermentation products, cell and micro-organism cultures and other samples in which the detection of an analyte, e.g., a multimeric analyte, is desirable. Samples can also include samples obtained from a subject and can include mucus, pus, blood, plasma, serum, semen, saliva, milk, stool swabs, sputum, bronchoalveolar lavage fluid, tissue samples, urine, spinal fluids, nasal fluids, etc. A “fluid sample” specifically refers to a non-solid sample. A “subject” from whom a sample is taken can be a human or a non- human animal (e.g., fish, amphibians, reptiles, birds, or mammals (e.g., rodents, dogs, cats, horses, cattle, sheep)). For example, a subject can be a mammalian subject, such 57#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 as, a human or a non-human veterinary subject (e.g., a cat, dog, rabbit, horse, goat, or pig). Next, the sample is contacted (e.g., mixed) with one or more (e.g. a plurality of) pairs of fusion proteins wherein each pair is specific (i.e., distinct or different) for one analyte, e.g., multimeric analyte and each pair comprises i) a first fusion protein comprising one or more binding domains fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding domains that bind to the same analyte as the first fusion protein fused to a second portion of the same reporter protein as the first fusion protein, wherein the first and second portions of the reporter protein, when together, comprise the reporter protein in its entirety, wherein the first fusion protein and / or the second fusion protein further comprises a fluorophore (preferably, wherein only one of the fusion proteins comprises a the fluorophore). Finally, the one or more analytes can be detected and identified based on mission from the fluorophore. The methods can include brining the fusion proteins into contact with a sample, and then detecting a signal from the fusion proteins. For reporter proteins that require a substrate, the methods include making a mixture comprising the substrate, the fusion proteins, and the sample, and then detecting a signal from the reporter. In some embodiments, the substrate can be provided in a mixture with the fusion proteins in a single container, e.g., in a well of a test strip. Alternatively, the substrate can be provided separately, and the sample can be added to the fusion proteins and then the substrate added, or the sample can be added to the substrate and then the mixed into the fusion proteins. In some embodiments, the sample can be added to the fusion proteins and then the substrate added, to allow sample and the fusion proteins to interact and then combine with the substrate, before the signal is detected. The emission from the fluorophore can occur within seconds or minutes (e.g., less than about 15 minutes, less than about 14 minutes, less than about 13 minutes, less than about 12 minutes, less than about 11 minutes, less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, less than about 1 minutes, less than about 60 seconds, less than about 50 seconds, less than about 40 seconds, less than about 30 seconds, less than about 20 seconds, less than about 15 seconds, less than about 10 58#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 seconds, less than about 9 seconds, less than about 8 seconds, less than about 7 seconds, less than about 6 seconds, less than about 5 seconds, less than about 4 seconds, less than about 3, seconds, less than about 2 seconds, or less than about 1 seconds. In some instances, the emission from the fluorophore occurs with milliseconds (e.g., less than about 1000 millisecond, less than about 900 millisecond, less than about 800 millisecond, less than about 700 millisecond, less than about 600 millisecond, less than about 500 millisecond, less than about 400 millisecond, less than about 300 millisecond, less than about 200 millisecond, less than about 100 millisecond, less than about 50 millisecond, less than about 25 millisecond, less than about 15 millisecond, less than about 10 millisecond, less than about 5 millisecond, less than about 4 millisecond, less than about 3 millisecond, less than about 2 millisecond, or less than about 1 millisecond. In an exemplary embodiment, if a sample comprising SARS-CoV-2, Influenza, and RSV is contacted with fusion proteins specific for these pathogenic viruses (i.e., the sample is contacted with three pairs of fusion proteins, one specific for SARS-CoV-2, Influenza, and RSV), the binding domains of the SARS-CoV-2 fusion proteins can bind to the RBD of SARS-CoV-2 spike protein (e.g., SARS-CoV- 2 wild-type spike), which can promote split luciferase complementation of the two luciferase fragments; for Influenza the trimeric structure on HA proteins promotes the split luciferase complementation of the two luciferase fragments; and for RSV, binding of the binding domains to the trimeric fusion glycoprotein (F) of RSV promotes the split luciferase complementation of the two luciferase fragments. When luciferin (a substrate of luciferase) is added, the functional luciferase oxidizes luciferin to produce an excited state oxyluciferin, which emits a photon. The emitted photon then excites one or more adjacent fluorescent acceptor(s). Excitation of the one or more adjacent fluorescent acceptor(s) allows for visual detection and identification of the pathogenic virus. The methods for detecting and identifying one or more analytes can include one fusion protein pair specific for SARS-CoV-2, one for Influenza, and one for RSV. Examples of fusion protein pairs are included in Table 3 below. 59#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Table 3: Exemplary pairs of fusion proteins: 60 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Expression Systems To use the fusion proteins described herein, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the fusion protein can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and / or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the fusion protein. The nucleic acid encoding the fusion protein can also be cloned into an expression vector, for administration to a plant cell, fungal cell, bacterial cell, protozoan cell, or animal cell, preferably a mammalian cell or a human cell. Thus, described herein are nucleic acid(s) encoding the fusion protein(s) described herein, vectors comprising the nucleic acid(s), and cells comprising the vector(s). In some embodiments, the vector is a lentivirus vector. See, e.g., Milone et al., “Clinical Use of Lentiviral Vectors,” Leukemia 32:1529–41 (2018). In some embodiments, the vector is a retrovirus vector. In some embodiments, the vector is a gamma retroviral vector. In some embodiments, the vector is a non-viral vector, e.g., a piggyback non-viral vector (PB transposon, see, e.g., Wu et al., “piggyback is a Flexible and Highly Active Transposon as Compared to Sleeping Beauty, Tol2, and Mos1 in Mammalian Cells,” PNAS 103(41):15008–13 (2006)), a sleeping beauty non-viral vector (SB transposon, see, e.g., Hudecek et al., “Going Non-Viral: the 61 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Sleeping Beauty Transposon System Breaks on Through to the Clinical Side,” Critical Reviews in Biochemistry and Molecular Biology 52(4):355–380 (2017)), or an mRNA vector. To obtain expression, a sequence encoding a fusion protein is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is selected from the group consisting of SV40, CMV, UBC, EF1A, PGK, and CAGG. In some embodiments, the promoter is an inducible promoter. See, e.g., Kallunki et al., “How to Choose the Right Inducible Gene Expression System for Mammalian Studies?” Cells 8:796 (2019). In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the fusion protein and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals. The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the fusion protein, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus 62#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells. Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983). Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the split fusion protein. In some embodiments, the cell(s) are stably transfected. In some embodiments, the cell(s) are transiently transfected. In some embodiments, the cell(s) are selected from the group consisting of HEK293T cells, Expi293, CAL51 cells, HCT116 cells, MCF7 cells, SKMEL28 cells, THP1 cells, U937 cells, and combinations thereof. In some embodiments, once the fusion proteins are expressed in and / or by a suitable system, the fusion proteins are purified. Purification of proteins are well known in the art. 63#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Variants In some embodiments, the fusion protein(s) or components thereof described herein, or the polynucleotides encoding the fusion protein(s) or components thereof described herein, are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., as described herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid "identity" is equivalent to nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher PlusTM, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign 64#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for target proteins or nucleic acids, the length of comparison can be any length, up to and including full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of the present disclosure, percent identity is relative to the full length of the query sequence. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences is accomplished using Smith Waterman Alignment with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Systems and Kits Also included herein are kits and systems (e.g., tool useful for diagnostic purposes) for use in a method described herein for detecting one, two, or more analytes in a sample. For example, the system or kit can comprise: A) a container for collecting a sample and B) a plurality of pairs of fusion proteins comprising pairs that are specific for one analyte. The plurality includes pairs that bind to a plurality of analytes, and can also include a number of pairs that bind to the same analyte. Each pair comprises i) a first fusion protein comprising one or more binding domains fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding domains that bind to the same analyte as the first fusion protein fused to a second portion of the same reporter protein as the first fusion protein. In some instances, the kit or system comprises at least three pairs of fusion proteins: one pair for SARS-CoV-2, one for Influenza, and one for RSV. In some instances, the one or more fusion proteins are provided in the kit or system in a composition, as described above. In some instances, the fusion proteins are lyophilized. The system or kit can also include directions (e.g., instructions) to be followed, e.g., by a subject or by a healthcare provider, e.g., to perform a method described herein. In some instances, not 65#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 all of the components of the system are not provided together. Alternatively, the components can be provided together in a kit, in a single package. The systems and kits can further comprise a substrate of the reporter protein. For example, luciferin or a derivative of luciferin can be present as substrate in a kit, wherein the fusion proteins comprise split luciferase fragments (e.g., small and large luciferase fragments). In some embodiments, the first and second fusion proteins in a system or kit is lyophilized. For example, a composition comprising one or more pairs of fusion proteins can be lyophilized for use in a system. A system or kit as described herein can comprise a negative control sample. The negative control sample can comprise 1-10% PBS, such as, 1-2%, 2-4%, 4-6%, 6- 8%, or 8-10% PBS (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% PBS). Additionally, or in the alternative, a system or kit described herein can comprise a positive control sample. A positive control sample can comprise the analyte or a component thereof that is known to specifically bind to the fusion proteins (e.g., the first and second fusion proteins) of the system and reconstitute the function of the reporter protein. In some instances, not all of the components of the system are provided together. Alternatively, the components can be provided together in a kit, in a single package. Table 4: Additional Sequences 66 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 67 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 68 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 69 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 70 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 71 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 72 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 73 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 74 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 75 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 76 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 77 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 78 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 79 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 80 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 81 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 82 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 83 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 84 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 85 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1: Multicolor MiniRGB probes for multiplexed assay Materials and Methods The study was approved by the Institutional Review Boards (IRBs) at Brigham and Women’s Hospital with the protocol number 2021B000047, and the procedures were in accordance with institutional guidelines. The positive and negative human saliva samples were purchased from Lee Biosolutions, Inc. Samples were de- identified before delivery to the lab and used without additional preprocessing. The following reagent was obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1 / 2020, Gamma-Irradiated, NR-52287, contributed by the Centers for Disease Control and Prevention; Influenza A Virus, A / California / 07 / 2009 (H1N1)pdm09, Egg Isolate (Produced in Eggs), NR-13663; Influenza B Virus, B / Brisbane / 60 / 2008 (Victoria Lineage), NR-42005; Spike Glycoprotein (Stabilized) from MERS Coronavirus, England 1 with C-Terminal Histidine and Twin-Strep® Tags, Recombinant from HEK293 Cells, NR-53591; Spike Glycoprotein (Stabilized) from SARS Coronavirus, Tor2 with C-Terminal Histidine and Strep® II Tags, Recombinant from HEK293 Cells, NR-53590; Hemagglutinin (HA) Protein from Influenza Virus, B / Ohio / 1 / 2005, Recombinant from Baculovirus, NR-19243; H7 Hemagglutinin (HA) Protein from Influenza Virus, A / Guangdong / 17SF003 / 2016 (H7N9), Recombinant from Baculovirus, NR-51203. SARS-CoV-2 BA.1 (Omicron) spike proteins were obtained from Bing Chen’s lab at Division of Molecular Medicine, Department of Pediatrics, Boston Children’s 86 #14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Hospital, Harvard Medical School. SARS-CoV-2 XBB (Omicron) spike proteins were obtained from Sino Biological US Inc. (U.S.A.) with the Cat # 40589-V08H40 and supplied as sterile 25 mM sodium citrate, 200 mM NaCl, 0.02% Tween 80, pH6.0. Bacterial expression and purification of recombinant proteins The DNA sequences of the fusion proteins were cloned into pET-28(a)+ or pET-26(b)+ for cytoplasmic and periplasmic protein expression, respectively. Plasmid constructs were reconstituted in nuclease-free water, and 10 ng of plasmid constructs were used to transform 10 ^L of competent E. coli cells according to Sambrook et al., 1989 (39). The E. coli cells were plated on LB agar plates supplemented with kanamycin in the concentration of 50 ^g / mL and incubated at 37 °C overnight to form bacterial colonies. Then, one single colony was picked into 10 ml of LB broth supplemented with 50 ^g / ml Kanamycin to make the starter cultures, which were grown overnight at 37 °C. On the next day, 10 mL of the starter culture was used to scale up 1 liter. Expression was induced at an OD600 of 0.6 with 1 mM sopropyl ^-d-1-thiogalactopyranoside (IPTG), and cells were grown for another 12-18 h at 20 °C. Cells were harvested by centrifugation (8,000 rpm, 20 minutes, 4 °C), and cell pellets were stored at -80 °C. For purification, cell pellets were resuspended in lysis buffer (50 mM Tris- HCl, pH 8, 500 mM NaCl, 1% Triton X-100) supplemented with a protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were sonicated on an ice bath for 20 minutes with 1-second-on and 3-second-off bursts. The lysate was centrifuged at 20,000 g for 30 minutes at 4 °C, and the supernatant was loaded onto a Ni-NTA gravity column. The column was washed with lysis buffer and then with washing buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0). Proteins were eluted with elution buffer (20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 8.0). The eluted proteins were further purified by size exclusion chromatography with Superdex 200 Increase 10 / 300 GL or HiLoad 16 / 600 Superdex 75 pg columns equilibrated with PBS. Mammalian expression and purification of recombinant proteins All constructs were synthesized by GenScript and cloned into the pcDNA3.4 expression vector. Protein expression was performed in Expi293 cells using the 87#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 ExpiFectamine™ 293 Transfection Kit (Thermo Fisher, Cat. No. A14525), following the manufacturer’s protocol. Briefly, Expi293 cells were seeded in a 24-deep-well plate (NEST, Cat. No. 51001) at a density of 3 × 10^ cells / ml. For transfection, 1^µg of plasmid DNA was diluted in 150^µl of Opti-MEM™ Reduced Serum Medium in one microcentrifuge tube, while 8^µl of ExpiFectamine™ 293 Reagent was diluted in 240^µl of Opti-MEM™ in a separate tube. After a 5-minute incubation, the contents of both tubes were combined and incubated for 20 minutes at room temperature before being added dropwise to the cells. Transfected cells were incubated in a shaking incubator for 20 hours. On the following day, 15^µl of ExpiFectamine™ 293 Transfection Enhancer 1 and 150^µl of Enhancer 2 were added dropwise to each well. The cells were then maintained in the shaking incubator for an additional six days. After incubation, the cell suspension was centrifuged at 500^rpm for 5 minutes to pellet the cells, and the supernatant was collected. The pH of the supernatant was verified to be approximately 7.0, and 50^µl of cOmplete His-Tag Purification Resin (Roche, Cat. No. 64-17-5) was added. The mixture was incubated overnight on a shaker at 4^°C to allow binding. Proteins were subsequently purified using a gravity-flow column. The resin was washed with five column volumes (CV) of PBS containing 10^mM imidazole and eluted with three CV of PBS containing 500^mM imidazole. The purity of the eluted proteins was assessed by SDS-PAGE followed by Coomassie blue staining. Expression and purification of the spike protein SARS-CoV-2 spike ectodomain (S.dTM.PP) with deletion of the furin cleavage site, two proline mutations, a foldon trimerization domain and a C terminal His tag was used in our study. The protein was stably expressed in Expi293 cells. Protein was purified from filtered cell supernatants using NiNTA resins before being subjected to additional purification by size-exclusion chromatography using Superdex 200 Increase 10 / 300 GL or Superose® 6 Increase 10 / 300 (GE Healthcare) equilibrated with PBS. Binding affinity analysis of B3L / B3S using biolayer interferometry (BLI) Binding of B3L / B3S to SARS-CoV-2 spike proteins was measured using a Fortebio Octet RED384 system (ForteBio). SARS-CoV-2 spike proteins were diluted 88#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 using the running buffer (PBST: 1xPBS, 0.05% Tween 20) and transferred to a 96- well plate. The Fortebio Octet RED384 instrument was used at 30 ^ with shaking at 1,000 rpm. Octet® AR2G biosensors (Fortebio, Cat# 18-5092) were used in this assay. The BLI assay was performed as described below: (1) Hydration: sensors were hydrated in 1x PBS for 5 minutes. (2) Activation: sensors were immersed in 20 mM EDC and 10 mM NHS for 10 min. (3) Loading B3L / B3S: sensors were immersed in 5 ^g / ml of B3S or 9 ^g / ml of B3L in 10 mM Acetate (pH = 4.5) for 30 seconds with around 1 nm of the binding signal increase. (4) Quench: Sensors were immersed in quenching buffer (100 mM Tris, 100 mM NaCl) for 10 minutes. (5) Baseline: sensors were immersed in PBST buffer for 5 minutes. (6) Association: sensors were immersed in SARS-CoV-2 spike proteins at different concentrations for 10 minutes. (7) Dissociation: sensors were immersed in PBST buffer for 50 minutes. Control sensors without B3L / B3S immobilization were also dipped in SARS- CoV-2 spike solutions and PBST as references. Recorded sensorgrams with background subtracted from the references were analyzed using the software Octet Data Analysis HT version 12.0 (ForteBio). All the curves were fit to a 1:1 binding model. Binding affinity analysis of L84 / S84 using microscale thermophoresis (MST) The binding affinity between L84 / S84 and influenza HA proteins was measured using MST. The measurements were performed on a Monolith NT.115 system (NanoTemper Technologies). We measured the fluorescence signal of L84 / S84 by using the Monolith His-Tag labeling kit RED-tris-NTA 2nd generation kit (NanoTemper Technologies). The samples were prepared in PBS buffer containing 0.05% Tween 20. The concentration of L84 / S84 was constant at 20 nM. Influenza HAs were titrated in two-fold dilution steps. For measurements, the samples were filled into Monolith Series capillaries (Cat #: MO-K022). Measurements were performed at 2% light-emitting diode (LED) and 20% MST power with 30-second laser on and 5-second laser off. Fluorescence was excited at 89#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 605–645 nm, and emission was detected at 680–685 nm. MST measurement was repeated three times, and the results were analyzed using MO Affinity Analysis software (NanoTemper Technologies). Gel analysis of spike-B3L-B3S complex 100 ^l of B3S (9 ^M) and 100 ^l of B3L (18 ^M) were mixed and incubated for 5 minutes. Then, 100 ^l of wild-type spike (5 ^M) was added into the B3S / B3L mixture and incubated for 30 minutes to form the spike-B3L-B3S complex. Next, the complex was purified by size exclusion chromatography (SEC, Superdex 200 Increase 10 / 300 GL, GE Healthcare, equilibrated in 1X PBS). The fractions corresponding to the spike-B3L-B3S complex were pooled and then subjected to a second SEC round. Finally, the fractions corresponding to the spike-B3L-B3S complex from the second SEC were concentrated using Amicon centrifugal filters 100 KDa MWCO (MilliporeSigma) and then assessed using SDS-PAGE. Bioluminescence measurements of SARS-CoV-2 spikes and viruses For the analysis of SARS-CoV-2 spikes, the assay was performed in a black 96-well plate, and bioluminescence was measured using a plate reader. 10 µl of B3L and 10 µl of B3S were mixed, followed by the addition of 10 µl of SARS-CoV-2 spikes at varying concentrations. The resulting solution was incubated at room temperature for 10 minutes. Then, 70 µl of luciferase substrate (40 µM, Nano-Glo® In Vivo Substrate, Promega) was added, and bioluminescence was quantified after 5 minutes using the plate reader (GloMax Discover, Promega) with a 0.5-second integration time. The signal-to-noise ratio at various concentrations of B3L and B3S was evaluated to optimize assay performance, with a final concentration of 1.5 nM for each applied in the optimized assay. For the analysis of Gamma-irradiated SARS-CoV-2 viruses spiked into the saliva and for clinical saliva samples from COVID-19 patients, the assay was performed in a black 24-well plate. 50 µl of B3L and 50 µl of B3S were mixed, followed by the addition of 100 µl of saliva sample and the mixture was incubated at room temperature for 10 minutes. To reduce potential inhibition of the luciferase reaction by interfering substances present in saliva, 1800 µl of luciferase substrate (40 90#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 µM, Nano-Glo® In Vivo Substrate, Promega) was added instead of 70 µl. Bioluminescence was then quantified after 5 minutes using the GloMax Discover plate reader with a 0.5-second integration time. Bioluminescence measurements of influenza HA proteins and viruses For the analysis of influenza HA proteins and viruses, the assay was performed in a black 96-well plate. 10 µl of L84 and 10 µl of S84 were mixed, followed by the addition of 10 µl of influenza HA proteins or viruses at varying concentrations. The mixture was incubated at room temperature for 10 minutes. Then, 70 µl of luciferase substrate (40 µM, Nano-Glo® In Vivo Substrate, Promega) was added, and the bioluminescence was quantified after 5 minutes using a plate reader (GloMax Discover, Promega) with a 0.5-second integration time. The signal-to-noise ratio at various concentrations of L84 and S84 was evaluated to optimize assay performance, with a final concentration of 3 nM for each applied in the optimized assay. Negative-stain electron microscopy and 3D reconstruction of B3L / B3L-Spike complex To form the SARS-CoV-2 spike protein-B3L / B3S complex, B3L and B3S were first mixed at a molar ratio of 3:5 and incubated at 4 ^ for 5 minutes, and then mixed with the spike protein at a final ratio of 3:5:2 and incubated at room temperature for 1 hour. The complex was purified by gel filtration chromatography on a Superose 610 / 300 column (GE Healthcare, Chicago, IL) equilibrated in 25 mM Tris-HCl, 150 mM NaCl, pH 7.5 buffer. For negative stain, 4 ^l of the complex was applied to a glow-discharged grid with a continuous carbon layer (Electron Microscopy Science) for 1 minute, and then stained with 2g / dL uranyl formate for 1 minute and blotted and air-dried. Grids were examined on a T12 electron microscope operating at 120 kV and nominal magnification of 62,000x, and ~100 images were collected on a CCD camera at 1.68 Å / pixel. Images were analyzed by 2D class classification, 3D classification and reconstruction using standard protocols with Relion 3.1.1 (40). Four classes were obtained and one of them clearly showed extra density for B3L / B3S with 6,067 particles. 3D reconstruction gave a final 3D density map at a resolution of 22 Å. We used the SARS-CoV-2 spike in complex with 91#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 miniprotein LCB3 structure (PDB ID: 7JZN) (32) and the NanoLuc Luciferase structure (PDB ID: 5IBO) for model fitting. The UCSF Chimera (41) was first used for rigid body fitting and Molecular Dynamics Flexible Fitting (MDFF) (42) was then used for further refinement. Structural biology applications used in this project were compiled and configured by SBGrid43. AlphaFold structure prediction and visualization AlphaFold was employed to predict the protein structures33,44. We performed the computation using ChimeraX which ran it on Google Colab servers and visualized the protein structures using PyMOL. Cryo-EM sample preparation and data collection Cryo-EM single particle analysis was carried out following the detailed protocols published previously (45). Briefly, to prepare cryo EM grids, the purified soluble spike protein34, B3S, and B3L were mixed at a molar ratio of 1:3:4.8, and the complex was further purified by gel filtration chromatography. The complex at 1.76 mg / ml was applied to a 1.2 / 1.3 Quantifoil gold grid (Quantifoil Micro Tools GmbH), which had been glow discharged with a PELCO easiGlowTM Glow Discharge Cleaning system (Ted Pella, Inc.) for 60 seconds at 15 mA. Grids were immediately plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific), and excess protein was blotted away using grade 595 filter paper (Ted Pella, Inc.) with a blotting time of 4 s, a blotting force of -12 at 4 °C with 100% humidity. The grids were first screened for ice thickness and particle distribution. Selected grids were used to acquire images with a Titan Krios transmission electron microscope (ThermoFisher Scientific) operated at 300 keV and equipped with a BioQuantum GIF / K3 direct electron detector. Automated data collection was carried out using SerialEM version 4.0.5 (46) at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.83 Å) at an exposure rate of 13.44 electrons per pixel per second. Each movie adds a total accumulated electron exposure of ~50.66 e- / Å2, fractionated in 47 frames. Data sets were acquired using a defocus range of 0.5-2.2 ^m. 92#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Image processing and 3D reconstructions All data were processed using cryoSPARC v.3.3.147. Drift correction for cryo-EM images was performed using patch mode, and contrast transfer function (CTF) was estimated by patch mode. Motion corrected sums with dose-weighting were used for all other image processing. Blob particle picking was then performed and 46,052,480 particles were extracted from 25,077 images using a box size of 672 Å (downsizing to 128 Å). The particles were subjected to ten rounds of 2D classification, giving 9,451,014 good particles. A low-resolution negative-stain reconstruction of the Wuhan-Hu-1 (D614) sample was low-pass filtered to 40Å resolution and used as an initial model. The selected good particles were first used for two rounds of heterogeneous classification with six copies of the initial model as a reference in C1 symmetry. Particles from a major class (37.9%) with clear structural features were re-extracted with a smaller box size (480Å) and subjected to another round of heterogeneous refinement with three copies of the initial model as the reference in C1 symmetry. Two major classes, representing two different two-RBD- up conformations, were obtained with class I containing 331,406 particles, and class II containing 270,437 particles. These two-RBD-up classes were then subjected to one round of duplicate removing to avoid over-refinement, then one round of CTF refinement and non-uniform refinement in C1 symmetry, giving a map at 3.3 Å resolution for class I and a map at 3.5Å resolution for class II. To further sort out potential heterogeneity, two rounds of heterogeneous refinement by cryoSPARC and one more round of focus-classification by Relion were carried out for each class and no new classes were obtained. To improve the local resolution at the interface between B3S / B3L and the RBD, several different local refinements were performed with different sizes of soft masks covering both the RBD and B3S / B3L. For the class I conformation, the local refinements gave a map at 3.4 Å resolution (for local refinement 1) and another map at 3.9 Å resolution. Likewise, the local refinements gave two 3.7 Å and 5 Å maps for the class II conformation. The difference between the two conformations is that class II RBDs are opened wider, perhaps more dynamic, than class I RBDs, possibly explaining the better density for the interface with the class I RBD. The best overall maps and the corresponding local maps using RBD- B3S / B3L masks were used together for model building. All resolutions were reported from the gold-standard Fourier shell correlation using the 0.143 criterion (36). 93#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Density maps were corrected from the modulation transfer function of the K3 detector and sharpened by applying a temperature factor that was estimated using Sharpening Tools in cryoSPARC. Local resolution was also determined using cryoSPARC. Model building The initial templates for the model building were our soluble S trimer structures (PDB ID:7KJ3) (34). We used AlphaFold to predict the structures of B3S and B3L independently. Several rounds of manual building were performed in Coot. The model was then refined in Phenix (48) against the 3.3 Å (class I), 3.5 Å (class II) cryo-EM maps. Iteratively, refinement was performed in both Phenix (real space refinement) and ISOLDE (49), and the Phenix refinement strategy included minimization_global, local_grid_search, and adp, with rotamer, Ramachandran, and reference-model restraints, using 7KJ3 as the reference models. The refinement statistics are summarized in Table 5, below. Structural biology applications used in this project were compiled and configured by SBGrid (43). Table 5. Cryo-EM statistics. 94#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Foldable plastic strip for lyophilized reagent storage and cellphone-based imaging A foldable plastic strip was designed for long-term reagent storage and cellphone-based luminescence imaging. The foldable plastic strip contains two caps on one end and two wells on the other end. The strip was printed by Ender-3 Pro 3D printer using a white polylactic acid (PLA) filament. The white interior of the wells enhanced the luminescence signal for cellphone-based detection. The external surface of the strip was covered by black sealing films (AbsorbMax, BK-50, Southern Labware) to avoid ambient light. 5% sucrose and 5% mannitol were used as cryoprotectants for reagent lyophilization. The strip containing the reagents was frozen at -80 °C for 24 hours, and then transferred into a precooled chamber of the freeze dryer (FreeZone 2.5, Labconco) for lyophilization. After 24 hours, the strip was removed from the freeze dryer. The lyophilized B3L / B3S was rehydrated using 1x PBS containing spike proteins, followed by folding the caps to cover the wells and rehydrate the substrate. Finally, the strip was fixed on the top of the front camera of 95#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 the cellphone (iPhone 13 pro, Apple) and the luminescence was imaged via a commercial application (Manual Cam). Statistical Analysis All the experiments in the study were repeated at least three times except for the analysis of clinical samples. Origin 9.1 software was used for graphical representation and statistical analyses. The error bars in the graphical data represent the means ± SDs. Statistical significance was determined using a Mann-Whitney test. A P value of <0.05 indicates statistical significance. The diagnostic metrics (i.e., sensitivity and specificity) were calculated using standard formulas. Data and materials availability All the relevant data are available from the corresponding author upon reasonable request. The atomic structure coordinates are deposited in the RCSB Protein Data Bank (PDB) under accession number 7KJ3. All materials generated during the current study are available from the corresponding author under a materials transfer agreement with Mass General Brigham. Calculation of BRET efficiency of MiniRGB probes For MiniRGB probes fused with bioluminescence protein and mNeonGreen, the BRET efficiency was calculated as where I525is the emission intensity at 525 nm (mNeonGreen), I455 is the emission intensity at455 nm (bioluminescence protein), and ^^ ^^^^^^^^^^ ^^^^ ratio of the bioluminescentemission intensity at 525 nm to the bioluminescent emission intensity at 455 nm from the spectrum of MiniRGB probes. We introduced ^ to correct the contribution of a small fraction of bioluminescent emission at the mNeonGreen emission wavelength. For MiniRGB probes fused with bioluminescence protein and LSSmOrange, the BRET efficiency was calculated as where I572is the emission intensity at 572 nm (LSSmOrange), I455 is the emission intensity at 455nm (bioluminescence protein), and ^^ ^^^^^^^^^^ ^^^^ ratio of the bioluminescentemission intensity at 572 nm to the bioluminescent emission intensity at 455 nm from 96#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 the spectrum of MiniRGB probes. We introduced ^ to correct the contribution of a small fraction of bioluminescent emission at the LSSmOrange emission wavelength. For MiniRGB probes fused with bioluminescence protein and CyOFP1, the BRET efficiency was calculated as (^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^×100%, where I589is the emission intensity at 589 nm (CyOFP1), I455is the emission intensity at 455 nm(bioluminescence protein), and ^^ ^^^^^^^^^^ ^^^^ ratio of the bioluminescent emissionintensity at 589 nm to the bioluminescent emission intensity at 455 nm from the spectrum of MiniRGB probes. We introduced ^ to correct the contribution of a small fraction of bioluminescent emission at the CyOFP1 emission wavelength. For MiniRGB probes fused with bioluminescence protein and mCyRFP1, the BRET efficiency was calculated as where I592 is the emission intensity at 592 nm (mCyRFP1), I455is the emission intensity at 455 nm(bioluminescence protein), and ^^ ^^^^^^^^^^ ^^^^ ratio of the bioluminescent emissionintensity at 592 nm to the bioluminescent emission intensity at 455 nm from the spectrum of MiniRGB probes. We introduced ^ to correct the contribution of a small fraction of bioluminescent emission at the mCyRFP1 emission wavelength. Results Since most viral surface proteins assemble as homo-multimers or hetero- multimers in close proximity, MiniRGB probes enable multicolor-based multiplexed detection of different viruses in a single reaction by genetically pairing various fluorescence proteins and proper binders targeting the homo-multimeric or hetero- multimeric surface proteins of different viruses, thus creating a color-based signal for identifying virus types (as used herein, color can mean non-visual wavelength lights of different wavelengths). To demonstrate this, we designed multicolor MiniRGB probes for the multiplexed detection of SARS-CoV-2, influenza, and RSV (FIG. 25A and Table 2 and Table 4, above), all of which lead to similar early symptoms. Binders with different affinities to the trimeric assembly of viral surface proteins with different proximity, as well as fluorescence proteins with different spectra overlap and different molecular brightness, are employed for designing multicolor MiniRGB probes to comparatively investigate their importance on BRET efficiency (FIGS. 26 and 27). The mNeonGreen and CyOFP1 are selected as the green and red 97#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 fluorescence proteins, respectively, due to their high molecular brightness and maximum overlap between the fluorescent excitation spectra and bioluminescent spectra (see, Table 6 below). The mNeonGreen is fused with the small luciferase fragment and B3 to form GSB3 for detecting SARS-CoV-2 spike proteins with the emission of green light (FIG. 25B). The CyOFP1 is fused with the small luciferase fragment and the antibody MEDI8897 to form RSMED which targets the trimeric assembly of respiratory syncytial virus (RSV) fusion (F) proteins with the emission of red light15. SEC and SDS-PAGE analysis confirm the high purity and the correct sizes of multicolor MiniRGB probes (FIG. 25B and FIG. 28, panels g and h). The L84 and S84 are utilized to detect the influenza HA proteins with the emission of blue light. The emission spectra were first measured to identify different types of viruses using individual donor-acceptor pairs of multicolor MiniRGB probes. The binding of L84 and S84 to the trimeric structure of influenza HA results in blue bioluminescence with the emission peak at 455 nm (FIG. 25C). Conversely, when B3L and GSB3 bind to the trimeric structure of SARS-CoV-2 spike proteins, energy transfer from bioluminescent donors to green fluorescent acceptors occurs, thereby generating emission with a green color peak at 525 nm (FIG. 25C). According to the calculation from the spectral data using the ratiometric intensity method (see above, “Calculation of BRET efficiency of MiniRGB probes”), B3L and GSB3 feature a BRET efficiency up to 95% in the presence of SARS-CoV-2 spike proteins. Similarly, the binding of LMED and RSMED to the trimeric structure of RSV F proteins leads to energy transfer from bioluminescent donor to red fluorescent acceptor with emission peaks at both 455 nm and 589 nm, leading to the emission with a pink color appearance (FIG. 25C). As shown in FIG. 27 and FIG. 29, higher affinity of binders, closer proximity between the trimeric assembly of viral surface proteins, maximized spectra overlap, and higher brightness generally contribute to a higher BRET efficiency. Altering the order of the protein domain also causes variations in the BRET efficiency, total emission intensity, and signal-to-noise ratio (FIGS. 25D and 25E, FIG. 29 and FIG. 30), underscoring the importance of sequence optimization. Different pairs of multicolor MiniRGB probes were combined to achieve multiplexed detection of viruses in a single reaction. The presence of both influenza HA proteins and SARS-CoV-2 spike proteins leads to the generation of cyan color with two emission peaks at both 455 nm and 525 nm (FIG. 5C). In contrast, the 98#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 presence of both SARS-CoV-2 spike proteins and RSV F proteins results in the emission of yellow color with three emission peaks at 455 nm, 525 nm, and 589 nm (FIG. 5G). We then examine the capability of multicolor MiniRGB probes for multiplexed detection of viral surface proteins in the nasal wash. The combination of B3L-GSB3 and L84-S84 pairs realizes the multiplex detection of SARS-CoV-2 spike proteins with a detection limit of 20 fM by monitoring the green color channel (FIG. 5F), and influenza HA proteins with a detection limit of 2.3 pM by monitoring the blue color channel in the same well (FIG. 5G). 99#14385889v1 %(:g p g calrA ep Sev o5.28. ;71.17.7dl4 5 7 7eiym w)aM Dk(6.2 4 2utna67.64.64.6 u 2 2 2 2 q:eY miQte;tfi1nL)sn(1.37.6. e- 3 3iciffeo o itcaruta )nonii0 8tM nm3 5c(n 1 1 - 1itxgeg:C A - m m m E. .*a)s / e7.7.6 5 grb K oop 5 5.5.r5.epBssaGsebRintpf.hg 5 wniiMrB8.2 g 94.325.714.w 03w(eniYsn8 4abgiQ.0.0 56.0 57.0 6 PseFd1-)mr00orf reofsC1-mc0 0 6,0 00 00 d 10,20,70,etmnietEM(1 5 2 04alonoorucmp s:ee )lakcdm5;thnmcot1 3 6 2 gan(iS 1 1 6 9needecwts ) rpero7 2 4 9almmeo 1 7 9 8dnau(u ^ 5 5 5 5clf eelfrao6 7 8 7osmxme0 3 2 9ry:nr e ( )^ 5 4 5 4atwem Mema ;nmegrnuane1rSopia1rP.v1tGe a5FOn P63h g 5eR Ftome5lee ry 1llS Ob 4mgaN C 4ya S A 1gaģ T N m L m C * 5 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 References 1. Baker, R.E. et al. Infectious disease in an era of global change. Nat. Rev. Microbiol., 1-13 (2021). 2. Morens, D.M., Folkers, G.K. & Fauci, A.S. Emerging infections: a perpetual challenge. Lancet Infect. Dis. 8, 710-719 (2008). 3. Davies, N.G. et al. Age-dependent effects in the transmission and control of COVID-19 epidemics. Nat. Med. 26, 1205-1211 (2020). 4. Callender, L.A. et al. The impact of pre-existing comorbidities and therapeutic interventions on COVID-19. Front. Immunol., 1991 (2020). 5. Yang, S. & Rothman, R.E. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect. Dis. 4, 337-348 (2004). 6. Kevadiya, B.D. et al. Diagnostics for SARS-CoV-2 infections. Nature materials, 1-13 (2021). 7. Boum, Y. et al. Performance and operational feasibility of antigen and antibody rapid diagnostic tests for COVID-19 in symptomatic and asymptomatic patients in Cameroon: a clinical, prospective, diagnostic accuracy study. Lancet Infect. Dis. 21, 1089-1096 (2021). 8. Larremore, D.B. et al. Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening. Sci. Adv. 7, eabd5393 (2021). 9. Peeling, R.W., Heymann, D.L., Teo, Y.-Y. & Garcia, P.J. Diagnostics for COVID-19: moving from pandemic response to control. The Lancet (2021). 10. Seo, G. et al. Rapid detection of COVID-19 causative virus (SARS-CoV-2) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor. ACS nano 14, 5135-5142 (2020). 11. Wang, L. et al. Rapid and ultrasensitive electromechanical detection of ions, biomolecules and SARS-CoV-2 RNA in unamplified samples. Nat. Biomed. Eng., 1- 10 (2022). 12. Broughton, J.P. et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. 38, 870-874 (2020). 13. Fozouni, P. et al. Amplification-free detection of SARS-CoV-2 with CRISPR- Cas13a and mobile phone microscopy. Cell 184, 323-333. e329 (2021). 101#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 14. Alafeef, M., Moitra, P., Dighe, K. & Pan, D. RNA-extraction-free nano- amplified colorimetric test for pointof-care clinical diagnosis of COVID-19. Nat. Protoc. 16, 3141-3162 (2021). 15. Huang, L. et al. One-step rapid quantification of SARS-CoV-2 virus particles via low-cost nanoplasmonic sensors in generic microplate reader and point-of-care device. Biosens. Bioelectron. 171, 112685 (2021). 16. Kobayashi, H., Picard, L.-P., Schönegge, A.-M. & Bouvier, M. Bioluminescence resonance energy transfer–based imaging of protein–protein interactions in living cells. Nat. Protoc. 14, 1084-1107 (2019). 17. Azad, T. et al. A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis. Nat. Commun. 9, 1-15 (2018). 18. Mezzanotte, L., van‘t Root, M., Karatas, H., Goun, E.A. & Löwik, C.W. In vivo molecular bioluminescence imaging: new tools and applications. Trends Biotechnol. 35, 640-652 (2017). 19. Iwano, S. et al. Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science 359, 935-939 (2018). 20. Su, Y. et al. Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nature Methods 17, 852-860 (2020). 21. Yeh, H.-W. et al. Red-shifted luciferase–luciferin pairs for enhanced bioluminescence imaging. Nature methods 14, 971-974 (2017). 22. Tannous, B.A. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat. Protoc. 4, 582-591 (2009). 23. Pfleger, K.D. & Eidne, K.A. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nature methods 3, 165-174 (2006). 24. Li, G., Wu, C., Ma, D.-L. & Leung, C.-H. Drug screening strategies using metal-based luminescent probes. TrAC Trends in Analytical Chemistry 139, 116270 (2021). 25. Xie, X. et al. A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19. Nat. Commun. 11, 1-11 (2020). 26. Elledge, S.K. et al. Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection. Nat. Biotechnol. 39, 928-935 (2021). 102#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 27. Quijano-Rubio, A. et al. De novo design of modular and tunable protein biosensors. Nature 591, 482-487 (2021). 28. Yao, Z. et al. A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies. Nat. Commun. 12, 1-8 (2021). 29. Kainulainen, M.H. et al. High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay. Sci. Rep. 11, 1-9 (2021). 30. Mercer, T.R. & Salit, M. Testing at scale during the COVID-19 pandemic. Nature Reviews Genetics 22, 415-426 (2021). 31. Suzuki, K. et al. Five colour variants of bright luminescent protein for real- time multicolour bioimaging. Nat. Commun. 7, 1-10 (2016). 32. Cao, L. et al. De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370, 426-431 (2020). 33. Torchia, J.A. et al. Optimized ACE2 decoys neutralize antibody-resistant SARS-CoV-2 variants through functional receptor mimicry and treat infection in vivo. Sci. Adv. 8, eabq6527 (2022). 34. Monteil, V. et al. Clinical grade ACE2 as a universal agent to block SARS^CoV^2 variants. EMBO Mol. Med. 14, e15230 (2022). 35. Ou, J. et al. ACE2-Targeting antibody suppresses SARS-CoV-2 Omicron and Delta variants. Signal transduction and targeted therapy 7, 1-3 (2022). 36. Xiao, T. et al. A trimeric human angiotensin-converting enzyme 2 as an anti- SARS-CoV-2 agent. Nat. Struct. Mol. Biol. 28, 202-209 (2021). 37. Dixon, A.S. et al. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem. Biol. 11, 400-408 (2016). 38. Cheong, J. et al. Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device. Nat. Biomed. Eng. 4, 1159–1167 (2020). 39. Laursen, N.S. et al. Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin. Science 362, 598-602 (2018). 40. Harris, A. et al. Influenza virus pleiomorphy characterized by cryoelectron tomography. Proceedings of the National Academy of Sciences 103, 19123-19127 (2006). 41. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual. (Cold spring harbor laboratory press, 1989). 103#14385889v1 Attorney Docket No. B0801.70396WO00 / BWH 2024-397 42. Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519-530 (2012). 43. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605-1612 (2004). 44. Trabuco, L.G., Villa, E., Schreiner, E., Harrison, C.B. & Schulten, K. Molecular dynamics flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods 49, 174-180 (2009). 45. Morin, A. et al. Cutting edge: Collaboration gets the most out of software. elife 2, e01456 (2013). 46. Mirdita, M. et al. c. Nature Methods, 1-4 (2022). 47. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021). 48. Cai, Y. et al. Distinct conformational states of SARS-CoV-2 spike protein. Science 369, 1586-1592 (2020). 49. Mastronarde, D.N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36-51 (2005). 50. Punjani, A., Rubinstein, J.L., Fleet, D.J. & Brubaker, M.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature methods 14, 290-296 (2017). 51. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D: Biological Crystallography 66, 213-221 (2010). 52. Croll, T.I. ISOLDE: a physically realistic environment for model building into low-resolution electrondensity maps. Acta Crystallographica Section D: Structural Biology 74, 519-530 (2018). OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 104#14385889v1

Claims

Attorney Docket No. B0801.70396WO00 / BWH 2024-397 WHAT IS CLAIMED IS:

1. A method for detecting and identifying the presence or absence of one or more analytes, e.g., multimeric analytes, optionally multimeric proteins expressed by pathogenic organisms, in a sample, the method comprising: a) providing a sample, optionally a sample from a subject or an environmental sample; b) contacting the sample with a plurality of pairs of fusion proteins wherein each pair of fusion protein is distinct for one analyte and each pair comprises i) a first fusion protein comprising one or more binding domains fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding domains that bind to the same analyte as the first fusion protein fused to a second portion of the same reporter protein as the first fusion protein, wherein the first and second portions of the reporter protein, when together, comprise the reporter protein in its entirety and restore its function, wherein the first fusion protein and / or the second fusion protein further comprises a fluorophore (preferably, wherein only one of the fusion proteins comprises a the fluorophore), and wherein the fluorophore for each pair has a distinguishable emission spectrum; and c) detecting and / or identifying one or more analytes based on the emissions from one or more of the fluorophores, optionally wherein detection occurs within 3 minutes or within 30-60 seconds.

2. The method of claim 1, wherein the one or more binding domains for each pair of the plurality of fusion proteins is specific for a distinct analyte, thereby making each pair of fusion protein specific for one analyte, optionally wherein the one or more binding domains are binders that recognize and bind at or near the apex of the distinct multimeric analyte, optionally to non-overlapping epitopes at or near the apex of the distinct multimeric analyte.

3. The method of 1 or 2, wherein the reporter protein is a luminescence-based enzyme. 105#14385889v1Attorney Docket No. B0801.70396WO00 / BWH 2024-397 4. The method of any one of claims 1 to 3, wherein the emission spectra of the reporter protein overlaps with the excitation spectra of the fluorophore.

5. The method of any one of claims 1 to 4, wherein the fluorophore is a small molecule fluorophore.

6. The method of any one of claims 1 to 5, wherein the fluorophore is fused either to the first fusion protein or the second fusion protein.

7. The method of any one of claims 1 to 6, wherein the first portion of the reporter protein and the second portion of the reporter protein are different sizes.

8. The method of claim 7, wherein the fluorophore is genetically fused to the fusion protein comprising a smaller portion of the reporter protein.

9. The method of any one of the preceding claims, wherein the fluorophore is different for each of the plurality of pairs of fusion proteins.

10. The method of any one of the preceding claims, wherein the reporter protein is luciferase.

11. The method of claim 10, wherein the substrate for the reporter protein is luciferin or a derivative of luciferin.

12. The method of claim 10, wherein the fluorophore is mNeonGreen, mCyRFP1, CyOFP1, and / or LSSmOrange.

13. The method of any one of claims 1 to 12, wherein step b) comprises contacting the sample with the plurality of pairs of fusion proteins under conditions effective for the reporter protein to generate a detectable signal. 106#14385889v1Attorney Docket No. B0801.70396WO00 / BWH 2024-397 14. The method of claim 13, wherein conditions effective for the reporter protein to generate a detectable signal comprises adding a composition comprising a substrate for the reporter protein.

15. The method of any one of the preceding claims, wherein the first portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO:

2.

16. The method of claim 15, wherein the second portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO:

1.

17. The method of any one of claims 1 to 14, wherein the first portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO:

3.

18. The method of claim 17, wherein the second portion of the reporter protein comprises at least about 80% sequence identity to SEQ ID NO:

4.

19. The method of any one of the preceding claims, wherein the first portion of the reporter protein is fused either to an N-terminal portion of the first fusion protein or to a C-terminal portion of the first fusion protein.

20. The method of any one of the preceding claims, wherein the second portion of the reporter protein is fused either to an N-terminal portion of the second fusion protein or to a C-terminal portion of the second fusion protein.

21. The method of any one of the preceding claims, wherein the first fusion protein further comprises a linker between the binding domain and the first portion of a reporter protein.

22. The method of any one of the preceding claims, wherein the second fusion protein further comprises a linker between the binding domain and the second portion of a reporter protein. 107#14385889v1Attorney Docket No. B0801.70396WO00 / BWH 2024-397 23. The method of any one of the preceding claims, wherein the first and second fusion proteins further comprise a purification tag.

24. The method of claim 23, wherein the purification tag is a FLAG tag, a c- Myc tag, a biotin tag, a histidine tag, or combination thereof.

25. The method of claim 24, wherein the purification tag is a histidine tag and / or c-Myc tag.

26. The method of any one of the preceding claims, wherein the first and / or second fusion proteins comprise one or more linkers between each component of the first and / or second fusion proteins.

27. The method of claim 26, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long.

28. The method of any one of the preceding claims, wherein the one or more analytes are multimeric proteins expressed by one or more viruses, bacteria, fungus, parasites, or mammalian cells, optionally wherein the viruses, bacteria, fungus, or parasites are pathogenic.

29. The method of claim 28, wherein the one or more pathogenic viruses comprises or consists of SARS-CoV-2, Influenza, and RSV.

30. The method of claim 28 or claim 29, wherein at least one pair of fusion proteins comprises a pair of fusion proteins for SARS-CoV-2.

31. The method of claim 30, wherein the pair of fusion proteins for SARS- CoV-2 is selected from the following: a) Histag-B3-nLuc(51-171) (SEQ ID NO: 27) and Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); b) Histag-B3-nLuc(51-171)-mNeonGreen (SEQ ID NO: 28) and 108#14385889v1Attorney Docket No. B0801.70396WO00 / BWH 2024-397 Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); c) i) His-B3-L (SEQ ID NO: 53) or ii) His-L-B3 (SEQ ID NO: 54) and i) Histag-B3-S-mNeonGreen (SEQ ID NO: 30) or ii) Histag- mNeonGreen-S-B3 (GSB3) (SEQ ID NO: 31); d) Histag-B3-L-mNeonGreen (SEQ ID NO: 32) and i) His-B3-S (SEQ ID NO: 51) or ii) His-S-B3 (SEQ ID NO: 52); e) His-L-I (SEQ ID NO: 56) and i) His-I-S-mNeonGreen (SEQ ID NO: 33), ii) His-I-mNeonGreen-S (SEQ ID NO: 34), or iii) His-Ihalf1- mNeonGreen-S-Ihalf2(SEQ ID NO: 35).

32. The method of any one of claims 28-31, wherein at least one pair of fusion proteins comprises a pair of fusion proteins for Influenza.

33. The method of claim 32, wherein the pair of fusion proteins for Influenza is selected from the following: i) His-84-L (SEQ ID NO: 74) or ii) L-84-His (SEQ ID NO: 75), and i) mCyRFP1-S-84-His (SEQ ID NO: 36), ii) S-mCyRFP1-84-His (SEQ ID NO: 37), iii) CyOFP1-S-84-His (SEQ ID NO: 38), iv) S-CyOFP1-84-His (SEQ ID NO: 39), v) LSSmOrange-S-84-His (SEQ ID NO: 40), or v) S- LSSmOrange-84-His (SEQ ID NO: 41).

34. The method of any one of claims 28-31, wherein at least one pair of fusion proteins comprises a pair of fusion proteins for RSV.

35. The method of claim 34, wherein the pair of fusion proteins for RSV is selected from the following: i) His-MED-L (SEQ ID NO: 49) or ii) His-L-MED (SEQ ID NO: 48), and i) MED-S-LSSmOrange-Histag (SEQ ID NO: 42), ii) Histag-LSSmOrange-S- MED (SEQ ID NO: 43), iii) MED-S-mCyRFP1-Histag (SEQ ID NO: 44), iv) Histag-mCyRFP1-S-MED (SEQ ID NO: 45), v) MED-S-CyOFP1-Histag (SEQ ID NO: 46), or v) Histag-CyOFP1-S-MED (RSMED) (SEQ ID NO: 47). 109#14385889v1Attorney Docket No. B0801.70396WO00 / BWH 2024-397 36. The method of any one of the preceding claims, wherein the method is done in a single well.

37. A system or kit for detecting one or more analytes, e.g., multimeric analytes, in a sample, wherein the system comprises: A) a container for collecting a sample and B) a plurality of pairs of fusion proteins comprising pairs is that each bind to one of a plurality of analytes, wherein each pair comprises i) a first fusion protein comprising one or more binding domains that bind to a analyte fused to a first portion of a reporter protein, and ii) a second fusion protein comprising one or more binding domains that bind to the same analyte as the first fusion protein fused to a second portion of the same reporter protein as the first fusion protein.

38. The system or kit of claim 37, wherein the one or more analytes are one or more multimeric proteins expressed by mammalian cells, or pathogenic viruses, parasites, fungus, or bacteria.

39. The system or kit of claim 38, wherein the one or more pathogenic viruses comprises or consists of SARS-CoV-2, Influenza, and RSV.

40. The system or kit of claim 38 or claim 39, wherein at least one pair of fusion proteins comprises a pair of fusion proteins for SARS-CoV-2.

41. The system or kit of claim 40, wherein the pair of fusion proteins for SARS-CoV-2 is selected from the following: a) Histag-B3-nLuc(51-171) (SEQ ID NO: 27) and Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); b) Histag-B3-nLuc(51-171)-mNeonGreen (SEQ ID NO: 28) and Histag-B3-nLuc(6-50)-mNeonGreen (SEQ ID NO: 29); c) i) His-B3-L (SEQ ID NO: 53) or ii) His-L-B3 (SEQ ID NO: 54) and i) Histag-B3-S-mNeonGreen (SEQ ID NO: 30) or ii) Histag- mNeonGreen-S-B3 (GSB3) (SEQ ID NO: 31); d) Histag-B3-L-mNeonGreen (SEQ ID NO: 32) and i) His-B3-S (SEQ ID NO: 51) or ii) His-S-B3 (SEQ ID NO: 52); 110#14385889v1Attorney Docket No. B0801.70396WO00 / BWH 2024-397 e) His-L-I (SEQ ID NO: 56) and i) His-I-S-mNeonGreen (SEQ ID NO: 33), ii) His-I-mNeonGreen-S (SEQ ID NO: 34), or iii) His-Ihalf1- mNeonGreen-S-Ihalf2(SEQ ID NO: 35).

42. The system or kit of any one of claims 37-41, wherein at least one pair of fusion proteins comprises a pair of fusion proteins for Influenza.

43. The system or kit of claim 42, wherein the pair of fusion proteins for Influenza is selected from the following: i) His-84-L (SEQ ID NO: 74) or ii) L-84-His (SEQ ID NO: 75), and i) mCyRFP1-S-84-His (SEQ ID NO: 36), ii) S-mCyRFP1-84-His (SEQ ID NO: 37), iii) CyOFP1-S-84-His (SEQ ID NO: 38), iv) S-CyOFP1-84-His (SEQ ID NO: 39), v) LSSmOrange-S-84-His (SEQ ID NO: 40), or v) S- LSSmOrange-84-His (SEQ ID NO: 41).

44. The system or kit of any one of claims 37-43, wherein at least one pair of fusion proteins comprises a pair of fusion proteins for RSV.

45. The system or kit of claim 44, wherein the pair of fusion proteins for RSV is selected from the following: i) His-MED-L (SEQ ID NO: 49) or ii) His-L-MED (SEQ ID NO: 48), and i) MED-S-LSSmOrange-Histag (SEQ ID NO: 42), ii) Histag-LSSmOrange-S- MED (SEQ ID NO: 43), iii) MED-S-mCyRFP1-Histag (SEQ ID NO: 44), iv) Histag-mCyRFP1-S-MED (SEQ ID NO: 45), v) MED-S-CyOFP1-Histag (SEQ ID NO: 46), or v) Histag-CyOFP1-S-MED (RSMED) (SEQ ID NO: 47). 111#14385889v1

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