Methods, kits, and systems for detecting analyte level

Abstract

The present disclosure relates to a method for detecting an analyte level in a sample including providing a first RNA polymerase fragment comprising a first analyte affinity domain bound thereto at an end of the first RNA polymerase fragment, the first analyte affinity domain configured to bind to a target analyte; providing a second RNA polymerase fragment comprising a second analyte affinity domain bound thereto at an end of the second RNA polymerase fragment, the second analyte affinity domain configured to bind to a target analyte; providing a biological sample comprising a target analyte; adding the biological sample to the first RNA polymerase fragment and second RNA polymerase fragment to form a reaction mixture; and subjecting the reaction mixture to a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output, along with associated kits, systems, and platforms.

Description

METHODS, KITS, AND SYSTEMS FOR DETECTING ANALYTE LEVELCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of 63 / 622,682, filed on January 19, 2024, which is incorporated herein by reference in its entirety as if fully set forth below.GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under R01EB034301 awarded by the National Institutes of Health and 1R01EB022592, awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a sequence listing, which is submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 16, 2025, is named “011529 114716_Sequence Listing.xml” and is 121,004 bytes in size.FIELD OF THE DISCLOSURE

[0004] The various embodiments of the present disclosure relate generally to medical diagnostics, and more particularly, to methods, kits, and systems for detecting analyte levels.BACKGROUND

[0005] Diagnosis at the earliest manifestations of disease is often critical in disease management and treatment at both the individual patient and public health levels. Unfortunately, most gold standard clinical diagnostic tests — including high performance liquid chromatography (HPLC), mass spectrometry, reverse transcriptase polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assays (ELISA) — require expensive equipment and trained technicians working in a well-funded laboratory setting. The costly nature of thesetechniques impedes sufficiently broad public access, especially in low-resource areas with little access to high-quality healthcare and in many low-income countries where annual per capita healthcare expenditures are less than US$100. Point-of-care (POC) diagnostic tools have a substantial impact on the management of both individual and public health by increasing test accessibility, which enables earlier detection of disease, more ideal treatment plans, and better prognoses. However, existing POC platforms like the lateral flow assay (LFA) are not simple to reconfigure to new targets due to the complex, iterative optimization of multiple interconnected design parameters that is required. Thus, engineering rapid, effective, and affordable POC tools is a dire need.

[0006] Cell-free expression (CFE) systems are a particularly promising platform poised to address current needs for POC diagnostic technology. These systems are composed simply of cellular lysate, substrates, and energy sources needed to execute transcription and translation from DNA templates in vitro. CFE systems can be lyophilized for long-term storage at ambient temperature, react robustly in small volumes of human sample matrices, can yield colorimetric outputs for equipment-free result interpretation, and cost only $0.02-$0.04 per 1 pL reaction. Numerous CFE biosensors have been engineered to detect nucleic acid, ion, and small molecule biomarkers, with particularly impressive modularity and versatility in detecting nucleic acid sequences.

[0007] However, these technologies neglect an important class of biomarker: proteins, which are gold standard biomarkers for many diseases and the target of over 100 FDA-approved diagnostic tests. Despite this critical importance, abilities for modular detection of proteins using CFE are far less mature.

[0008] Only a few efforts for modular protein sensing using CFE have been reported, all of which use aptamers (oligonucleotides that bind to a specific target with high affinity) to regulate gene expression upon protein recognition. Modular implementation of newly evolved or synthetic aptamers remains a challenge, though, with integration into existing sensing platforms typically done through costly and time-consuming trial-and-error experimentation. A recent plug-and-play DNA aptamer platform sought to address this issue in an in vitro transcription system, but relies on the aptamers forming a specific noncanonical DNA structure called a G-quadruplex, limiting the sensing space of the platform significantly. A new automated design process was also recently reported that converts arbitrary RNA aptamers into riboswitches for use in CFE biosensors. While this is a significant advance in the long-standingchallenge of riboswitch engineering, it requires users to know the aptamer’s secondary structure when bound to the antigen and the antigen’s free energy of binding, specifications that are particularly challenging for new protein targets and thus impede its more widespread use. Critically, there currently are no aptamer-based CFE biosensors that have been linked to visual colorimetric outputs, tested in human samples, or lyophilized for storage and transportation, rendering these systems unsuitable for POC use.BRIEF SUMMARY

[0009] A first aspect of the present disclosure provides a method for detecting an analyte level in a sample. The method includes providing a first RNA polymerase fragment comprising a first analyte affinity domain bound thereto at an end of the first RNA polymerase fragment, the first analyte affinity domain configured to bind to a target analyte; providing a second RNA polymerase fragment comprising a second analyte affinity domain bound thereto at an end of the second RNA polymerase fragment, the second analyte affinity domain configured to bind to a target analyte; providing a biological sample comprising a target analyte; adding the biological sample comprising the target analyte to the first RNA polymerase fragment and second RNA polymerase fragment to form a reaction mixture; and subjecting the reaction mixture to a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output.

[0010] In any of the embodiments disclosed herein, the analyte is a protein, a glycan, a small molecule, a virus particle, or a combination thereof.

[0011] In any of the embodiments disclosed herein, the first analyte affinity domain is a protein affinity domain, or the second analyte affinity domain is a protein affinity domain, or both the first and second analyte affinity domains are protein affinity domains.

[0012] In any of the embodiments disclosed herein, the first RNA polymerase fragment and the second RNA polymerase fragment are configured to have minimal spontaneous reassembly.

[0013] In any of the embodiments disclosed herein, when the first RNA polymerase fragment and the second RNA polymerase fragment are forcibly colocalized upon binding of the first and second affinity domains to the target analyte, thus driving a reassembly of the first RNA polymerase fragment and the second RNA polymerase fragment, turning on a reporter gene, and generating the measurable output.

[0014] In any of the embodiments disclosed herein, the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, mucus, lymph, interstitial fluid, cerebrospinal fluid, pus, breast milk, and amniotic fluid.

[0015] In any of the embodiments disclosed herein, the first fragment, second fragment, and biological sample are added into a cell-free expression mixture, the mixture comprising lysates of cells capable of executing transcription and translation.

[0016] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

[0017] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a nanobody.

[0018] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a monobody.

[0019] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a DARPin.

[0020] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

[0021] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a nanobody.

[0022] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a monobody.

[0023] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a DARPin.

[0024] In any of the embodiments disclosed herein, the first analyte affinity domain is selected from the group consisting of NB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

[0025] In any of the embodiments disclosed herein, the second analyte affinity domain is selected from the group consisting ofNB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

[0026] In any of the embodiments disclosed herein, the first analyte affinity domain and the second analyte affinity domain comprise minimally overlapping epitopes configured to allow the first analyte affinity domain and the second analyte affinity domain to bind simultaneously to the target analyte.

[0027] In any of the embodiments disclosed herein, the first RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

[0028] In any of the embodiments disclosed herein, the first RNA polymerase fragment comprises a split T7 RNA polymerase.

[0029] In any of the embodiments disclosed herein, the first RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPC.L2A.

[0030] In any of the embodiments disclosed herein, the second RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

[0031] In any of the embodiments disclosed herein, the second RNA polymerase fragment comprises a split T7 RNA polymerase.

[0032] In any of the embodiments disclosed herein, the second RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPC,L2A.

[0033] In any of the embodiments disclosed herein, the measurable output comprises absorbance, fluorescence, luminescence, or a combination thereof.

[0034] In any of the embodiments disclosed herein, the measurable output comprises absorbance, fluorescence, or luminescence at 580 nm.

[0035] In any of the embodiments disclosed herein, the method further includes providing a linker sequence in the first RNA polymerase fragment, the second RNA polymerase fragment, or in both the first and second RNA polymerase fragments.

[0036] In any of the embodiments disclosed herein, the linker sequence is between 5 and 30 amino acids in length.

[0037] In any of the embodiments disclosed herein, the linker sequence comprises the amino acid sequences SEQ ID NOS: 104-106.

[0038] In any of the embodiments disclosed herein, the first RNA polymerase fragment is lyophilized, the second RNA polymerase fragment is lyophilized, or both the first RNA polymerase fragment and the second RNA polymerase fragment are lyophilized.

[0039] A second aspect of the present disclosure provides a kit for detecting an analyte level in a sample. The kit includes a first RNA polymerase fragment comprising a first analyteaffinity domain bound thereto at an end of the first RNA polymerase fragment, the first analyte affinity domain configured to bind to a target analyte; and a second RNA polymerase fragment comprising a second analyte affinity domain bound thereto at an end of the second RNA polymerase fragment, the second analyte affinity domain configured to bind to a target analyte.

[0040] In any of the embodiments disclosed herein, the analyte is a protein, a glycan, a small molecule, a virus particle, or a combination thereof.

[0041] In any of the embodiments disclosed herein, the first analyte affinity domain is a protein affinity domain, or the second analyte affinity domain is a protein affinity domain, or both the first and second analyte affinity domains are protein affinity domains.

[0042] In any of the embodiments disclosed herein, the first RNA polymerase fragment and the second RNA polymerase fragment are mixed with a biological sample to form a reaction mixture.

[0043] In any of the embodiments disclosed herein, the reaction mixture is subject to a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output.

[0044] In any of the embodiments disclosed herein, the first RNA polymerase fragment and the second RNA polymerase fragment are configured to have minimal spontaneous reassembly when subject to said condition.

[0045] In any of the embodiments disclosed herein, when the first RNA polymerase fragment and the second RNA polymerase fragment are forcibly colocalized upon binding of the first and second affinity domains to the target analyte, thus driving a reassembly of the first RNA polymerase fragment and the second RNA polymerase fragment, turning on a reporter gene, and generating the measurable output.

[0046] In any of the embodiments disclosed herein, the first fragment, second fragment, and biological sample are added into a cell-free expression mixture, the mixture comprising lysates of cells capable of executing transcription and translation.

[0047] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

[0048] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a nanobody.

[0049] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a monobody.

[0050] In any of the embodiments disclosed herein, the first analyte affinity domain comprises a DARPin.

[0051] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

[0052] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a nanobody.

[0053] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a monobody.

[0054] In any of the embodiments disclosed herein, the second analyte affinity domain comprises a DARPin.

[0055] In any of the embodiments disclosed herein, the first analyte affinity domain is selected from the group consisting ofNB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

[0056] In any of the embodiments disclosed herein, the second analyte affinity domain is selected from the group consisting ofNB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

[0057] In any of the embodiments disclosed herein, the first analyte affinity domain and the second analyte affinity domain comprise minimally overlapping epitopes configured to allow the first analyte affinity domain and the second analyte affinity domain to bind simultaneously to the target analyte.

[0058] In any of the embodiments disclosed herein, the first RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

[0059] In any of the embodiments disclosed herein, the first RNA polymerase fragment comprises a split T7 RNA polymerase.

[0060] In any of the embodiments disclosed herein, the first RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPC.L2A.

[0061] In any of the embodiments disclosed herein, the second RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

[0062] In any of the embodiments disclosed herein, the second RNA polymerase fragment comprises a split T7 RNA polymerase.

[0063] In any of the embodiments disclosed herein, the second RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPC.L2A.

[0064] In any of the embodiments disclosed herein, the measurable output comprises absorbance, fluorescence, luminescence, or a combination thereof.

[0065] In any of the embodiments disclosed herein, the measurable output comprises absorbance, fluorescence, or luminescence at 580 nm.

[0066] In any of the embodiments disclosed herein, the kit further includes providing a linker sequence in the first RNA polymerase fragment, the second RNA polymerase fragment, or in both the first and second RNA polymerase fragments.

[0067] In any of the embodiments disclosed herein, the linker sequence is between 5 and 30 amino acids in length.

[0068] In any of the embodiments disclosed herein, the linker sequence comprises the amino acid sequences SEQ ID NOS: 104-106.

[0069] In any of the embodiments disclosed herein, the first RNA polymerase fragment is lyophilized, the second RNA polymerase fragment is lyophilized, or both the first RNA polymerase fragment and the second RNA polymerase fragment are lyophilized.

[0070] Conventional laboratory protein detection techniques are not suitable for point-of-care (POC) use because they require expensive equipment and laborious protocols, and existing POC assays suffer from long development timescales. Here, a modular cell-free biosensing platform for generalizable protein detection that is referred to herein as TLISA (T7 RNA polymerase-Linked ImmunoSensing Assay) is described, designed for extreme flexibility and equipment- free use. TLISA uses a split T7 RNA polymerase fused to affinity domains against a protein. The target antigen drives polymerase reassembly, inducing reporter expression. The platform is characterized, then its modularity is demonstrated by using 16 affinity domains against four different antigens with minimal protocol optimization. TLISA is shown to be suitable for POC use by sensing human biomarkers in serum and saliva with a colorimetric readout within one hour and by demonstrating functionality after lyophilization. Altogether, this technology has the potential to enable truly rapid, reconfigurable, modular, and equipment- free detection of diverse classes of proteins.

[0071] To address this gap in capabilities and in the literature, a strategy is devised combining small, target-specific protein-binding affinity domains with a split T7 RNA polymerase (T7RNAP) to create a modular, easily engineerable, field-deployable protein detection platform. While antibodies are widely used in laboratory assays (e.g., ELISA) and in POC LFAs as specific and sensitive affinity domains, they are challenging to implement efficiently in CFE biosensing platforms due to post-translational modifications and challenges in proper folding. Kim and Swartz, “Efficient Production of a Bioactive, Multiple Disulfide-Bonded Protein Using Modified Extracts of Escherichia Coli,” Biotechnology and Bioengineering 85:122-129 (2004) and Ryabova et al., “Functional Antibody Production Using Cell-Free Translation: Effects of Protein Disulfide Isomerase and Chaperones,” Nat Biotechnol 15:79— 84 (1997), both of which are hereby incorporated by reference in their entirety. Thus, focus on the use of nanobodies was chosen. Nanobodies are recombinant antigen-binding domains derived from the variable region of the unique immunoglobulins produced by camelids that lack light chains (Fridy et al., “A Robust Pipeline for Rapid Production of Versatile Nanobody Repertoires,” Nat Methods 11: 1253-1260 (2014), which is hereby incorporated by reference in its entirety); they are functionally similar to monoclonal antibodies but can be expressed easily in bacterial CFE systems (Pardee et al., “Portable, On-Demand Biomolecular Manufacturing,” Cell 167:248-259.el2 (2016), which is hereby incorporated by reference in its entirety). Nanobodies are feasible to engineer because they are small (ca. 15 kDa), highly stable, and typically robust to changes in their chemical environment and to fusions with diverse molecules. Yang and Shah, “Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics,” Frontiers in Oncology 10 (2020), which is hereby incorporated by reference in its entirety. Moreover, nanobodies can be rapidly evolved to bind to new targets through processes such as phage, yeast, or ribosome display (McMahon et al., “Yeast Surface Display Platform for Rapid Discovery of Conformationally Selective Nanobodies,” Nat Struct Mol Biol 25:289-296 (2018) and Chen et al., “A Cell-Free Nanobody Engineering Platform Rapidly Generates SARS-CoV-2 Neutralizing Nanobodies,” Nat Commun 12:5506 (2021), both of which are hereby incorporated by reference in their entirety), in contrast to the more expensive and complex development pipeline for new antibody development (Gieselmann et al., “Effective High-Throughput Isolation of Fully Human Antibodies Targeting Infectious Pathogens,” NatProtoc 16:3639-3671 (2021), which is hereby incorporated by reference in its entirety). Thus, the potential ease of creating affinity domains via in vitro evolution makes theTLISA approach generalizable beyond the existing set of affinity domains for known targets. The split T7RNAP to which is connected these affinity domains has one fragment that was evolved (T7RNAPNcv) to have minimal spontaneous reassembly in the absence of forced colocalization; that co-localization can be caused by binding to a target via affinity domains fused to the polymerase fragments (Pu et al., “Evolution of a Split RNA Polymerase as a Versatile Biosensor Platform,” Nat Chem Biol 13:432 438 (2017) and Pu et al., “Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors,” ACS Chem. Biol. 13:431— 437 (2018), both of which are hereby incorporated by reference in their entirety). Neither nanobodies nor a split T7RNAP are known to have been used for cell-free biosensing despite their broad utility having been demonstrated in in vivo applications.

[0072] By fusing nanobodies or other affinity domains to each of the split T7RNAP fragments, a biosensing platform has be created that is called TLISA (T7RNAP-Linked ImmunoSensing Assay), analogous to a sandwich ELISA but in a faster, more user-friendly, and easily engineerable format (FIG. 1A). In the presence of a target antigen, both nanobodies bind, driving reassembly of the polymerase fragments and inducing expression of a reporter protein. Designing the platform around a split RNAP (as opposed to other split reporter proteins such as luciferase or GFP) allows for taking advantage of the intrinsic amplification of input signal by transcription and translation, which can potentially lead to a more sensitive biosensor. Additionally, this choice provides additional flexibility to the platform since the reporter protein can be easily replaced by any gene under regulation of a T7 promoter, allowing for outputs that may provide more quantitative precision (e.g., fluorescent reporters) or more equipment- free use (e.g., P-galactosidase) as needed for a given application. Here, the modularity and utility of the TLISA platform is demonstrated, characterizing design considerations using eGFP and mCherry as model target antigens and then showing that the modular nature of TLISA enables rapid sensor development for clinically relevant proteins.

[0073] These and other aspects of the present disclosure are described in the Detailed Description below and the accompanying drawings. Other aspects and features of embodiments will become apparent to those of ordinary skill in the art upon reviewing the following description of specific, exemplary embodiments in concert with the drawings. While features of the present disclosure may be discussed relative to certain embodiments and figures, all embodiments of the present disclosure can include one or more of the features discussed herein. Further, while one or more embodiments may be discussed as having certain advantageousfeatures, one or more of such features may also be used with the various embodiments discussed herein. In similar fashion, while exemplary embodiments may be discussed below as device, system, or method embodiments, it is to be understood that such exemplary embodiments can be implemented in various devices, systems, and methods of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, specific embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0075] FIGS. 1A-1D provide a proof-of-concept eGFP TLISA biosensor, in accordance with some embodiments of the present disclosure. FIG. 1 A shows a general TLISA schematic. The T7RNAPNCV(blue) fused to a nanobody (green) and the T7RNAPC(red) fused to a separate nanobody should not reassemble in the absence of antigen. In the presence of antigen, both nanobodies bind and bring T7RNAPNCVand T7RNAPCin close proximity, driving reassembly. The reassembled T7RNAP then drives expression of LacZ (purple). LacZ converts the yellow pigment CPRG into purple CPR to generate a range of visually interpretable output colors. FIG. IB shows a genetic circuit of the proof-of-concept eGFP biosensor. T7RNAPNevfused to anti-eGFP LaG2 and T7RNAPCfused to anti-eGFP LaG14 were expressed from separate plasmids under a native E. coli promoter (PBAD). In the presence of eGFP, both LaG2 and LaG14 bind and bring T7RNAPNev and T7RNAPCin close proximity, driving reassembly and expression of LacZ off the third, T7-regulated, reporter plasmid. FIG. 1C shows absorbance readings over time at 37 °C to monitor the conversion of yellow CPRG to purple CPR in the presence of either protein buffer, a control protein (mCherry), or eGFP and 0.1 nM pT7LacZ. Despite some low background reassembly of T7RNAP, target-dependent reassembly and expression is clearly visible. FIG. ID shows absorbance values and corresponding pictures of visible reaction colors after 30 minutes of incubation at 37 °C with different concentrations of eGFP or control protein (mCherry), showing detection range. Lines and bars represent thearithmetic mean ± standard deviation (shaded area) of n=3 technical replicates (white triangles). Robustness to variation across lysate batches and to reactions on different days is shown in FIGS. 10A-10B.

[0076] FIGS. 2A-2C demonstrate TLISA is robust to different affinity domains, in accordance with some embodiments of the present disclosure. FIG. 2A shows TLISA genetic sensing circuit and ABC (Equation 1) values for all 36 combinations of six anti-eGFP NBs. FIG. 2B shows eGFP TLISA built from a NB (LaG2) and a MB (GS2). FIG. 2C shows eGFP TLISA built from a NB (LaG2) and a DARPin (3G86.32). 500 nM mCherry was used as the control protein in all reactions. All reactions contain 0.1 nM pT7LacZ. Lines represent the arithmetic mean ± standard deviation (shaded area) of n=3 technical replicates.

[0077] FIGS. 3A-3D show impacts of linker length and fusion termini on sensor performance, in accordance with some embodiments of the present disclosure. FIG. 3A shows absorbance data of T7RNAPNev-NBl / LaG2-T7RNAPc TLISAs with different linker lengths. Different shapes represent different linker lengths for T7RNAPNCV-NB 1. Each graph represents a different linker length for LaG2-T7RNAPc. FIG. 3B shows ABC values for the data presented in FIG. 3A. FIG. 3C shows genetic circuits indicating the varying locations of the NB on the T7RNAPNSVfragment and absorbance data for TLISA reactions with NB1 fused to either the N- (circles) or C-terminus (triangles) of T7RNAPNev used with LaG2-T7RNAPc. ABC values for the two sensors are displayed in the graph. FIG. 3D shows genetic circuits indicating the varying locations of the NB on the T7RNAPC,L2A fragment and absorbance data for TLISA reactions with LaG2 fused to either the N- (circles) or C-terminus (triangles) of T7RNAPC,L2A used with T7RNAPNsv-LaG19. ABC values for the two sensors are displayed in the graph. All reactions contained 0.1 nM pT7LacZ except for the C-terminal NB fusion data in 3D, which used 0.2 nM pT7LacZ. 500 nM mCherry was used as the control protein for all reactions. Symbols represent the arithmetic mean ± standard deviation (shaded area) of n=3 technical replicates.

[0078] FIGS. 4A-4B illustrate that TLISA is modular for detection of different protein targets, in accordance with some embodiments of the present disclosure. FIG. 4A shows genetic circuit for sensing mCherry and corresponding absorbance versus time data. FIG. 4B shows ABC values for all combinations of mCherry NBs. All reactions had 0.05 nM pT7LacZ. 200 nM eGFP was used as the control protein for all reactions. Lines represent the arithmetic mean ± standard deviation (shaded area) of n=3 technical replicates.

[0079] FIGS. 5A-5C demonstrate that TLISA can be easily re-engineered to create biosensors for clinically relevant protein biomarkers, in accordance with some embodiments of the present disclosure. FIG. 5A shows genetic circuit and absorbance versus time of a TLISA sensor to detect SARS-CoV-2 RBD using two NBs (VHHE and VHHV). FIG. 5B shows one NB and one DARPin (FSR22). FIG. 5C shows genetic circuit and absorbance versus time data of a TLISA sensor detecting transthyretin (TTR) using NB03 on both polymerase fragments. Both SARS-CoV-2 biosensors used 0.1 nM pT7LacZ. The TTR biosensor used 0.12 nM pT7LacZ. 500 nM mCherry was used as the control protein in all reactions. Lines represent the arithmetic mean ± standard deviation (shaded area) of n=6 replicates performed on different days.

[0080] FIGS. 6A-6F demonstrate that TLISA functions robustly in complex sample matrices at room temperature and after lyophilization, in accordance with some embodiments of the present disclosure. FIG. 6A is a schematic of TLISA protocol when sensing proteins in biological samples. FIG. 6B shows end-point absorbance values after 50 minutes for an mCherry TLISA in various concentrations of either pooled human serum or saliva with 1% v / v RNAse inhibitor. FIG. 6C shows absorbance values after 60 minutes in a SARS-CoV-2 RBD TLISA in either 0% or 20% v / v pooled human saliva with 1% v / v RNAse Inhibitor. FIG. 6D shows end-point absorbance values after 90 minutes of incubation at 25 °C showing room temperature detection of 150 nM of SARS-CoV-2 S protein in 20% v / v pooled human saliva. Reactions used 0.5 nM pT7LacZ. MERS-CoV S protein was used as an off-target protein control. FIG. 6E shows photos of n=3 SARS-CoV-2 S protein TLISA reactions after 90 minutes of incubation at 25 °C showing equipment-free interpretation of test results. FIG. 6F shows end-point absorbance values of a lyophilized SARS-CoV-2 RBD TLISA reaction 60 minutes after rehydration with either 0% or 20% v / v pooled human saliva, 0.5% v / v RNAse Inhibitor, and 0.75 nM pT7LacZ. eGFP was used as a control in FIG. 6B. mCherry was used as a control protein for FIGS. 6C and 6F. Bars represent the arithmetic mean ± standard deviation of n=3 technical replicates (white diamonds).

[0081] FIG. 7 shows visualization of the anti-eGFP NB epitopes, in accordance with some embodiments of the present disclosure. GFP molecule is shown in gray, and residues that interact with each NB are shown in purple. Binding residues for Nbl were determined by Kubala et al. (Kubala et al., “Structural and Thermodynamic Analysis of the GFP:GFP- Nanobody Complex,” Protein Science 19:2389-2401 (2010), which is hereby incorporated by reference in its entirety) and mapped onto PBD 2QU1. The remaining NBs were determinedby Fridy et al. (Fridy et al., “A Robust Pipeline for Rapid Production of Versatile Nanobody Repertoires,” Nat Methods 11: 1253-1260 (2014), which is hereby incorporated by reference in its entirety) and mapped onto PDB 1B9C. Illustrations were made using PyMol.

[0082] FIG. 8 determines visible limit of detection, in accordance with some embodiments of the present disclosure. 17 people were shown 10 different pairs of reaction photos (top) in randomized order and asked whether they thought the colors were the same or different. Plotted are the absorbance values at 580 nm (symbols, right y axis) and the difference in absorbance values (bars, left y axis) for each pair. Two of the pairs were the same exact color (right two pairs). The poll responses (bottom pie charts) indicate that pairs with greater differences in absorbance values are more universally distinguishable. With the exception of one pair of colors, most of the responses identified AA580 values greater than 0.233 as different colors (first data points). Two colors with lower A580 values (i.e., more yellow) are more visually distinguishable even with lower AA580 values.

[0083] FIG. 9 depicts the detection range and LOD of the T7RNAPNcv-LaG2 / LaG14-T7RNAPc TLISA, in accordance with some embodiments of the present disclosure. Absorbance data with increasing concentrations of eGFP or control protein show an LOD of 100 nM eGFP after one hour. All reactions had 0.1 nM pT7LacZ. mCherry was used as the control protein. Bars represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0084] FIGS. 10A-10B show that TLISA performance has minimal variation across different batches of crude lysate and different reaction days, in accordance with some embodiments of the present disclosure. FIG. 10A shows absorbance data for a T7RNAPNcv-NBl / LaG2- T7RNAPCTLISA using two different batches of in-house prepared crude lysate. FIG. 10B shows absorbance data for a T7RNAPNcv-NBl / LaG2-T7RNAPcTLISA using two different batches of in-house prepared crude lysate performed two weeks apart. Symbols represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0085] FIG. 11 shows absorbance data corresponding to the data in FIG. 2A, in accordance with some embodiments of the present disclosure. Columns represent different T7RNAPCNB fusions and rows represent different T7RNAPNSV NB fusions. Lines and shaded areas represent the arithmetic mean ± standard deviation of n=3 technical replicates. Consistent with Figures 2B and 2C, the purple curve is for 500 nM eGFP, the yellow curve is for 500 nM control protein (mCherry), and the gray curve is for the buffer-only control.

[0086] FIGS. 12A-12D illustrate TLISA performance when using either the WT T7RNAPC or the identified T7RNAPC mutations, in accordance with some embodiments of the present disclosure. FIG. 12A shows T7RNAPNcv-NBl / LaG2-T7RNAPceGFP TLISA with either (left) WT T7RNAPCor (right) mutated T7RNAPC. FIG. 12B shows T7RNAPNcv-NBl / LaG2- T7RNAPceGFP TLISA using the WT T7RNAPcwith a range of different pT7LacZ reporter plasmid concentrations. FIG. 12C shows T7RNAPNcv-LaG2 / GS2-T7RNAPceGFP TLISA with either (left) WT T7RNAPCor (right) mutated T7RNAPC. FIG. 12D shows T7RNAPNev- LaG2 / 3G86.32-T7RNAPceGFP TLISA with either (left) WT T7RNAPCor (right) mutated T7RNAPC.

[0087] FIG. 13 depicts direct ELISA validation of anti-eGFP NB expression and functionality in a CFE system, in accordance with some embodiments of the present disclosure. Anti-eGFP NBs were translationally fused to LacZ. TTR was used as the control protein. Bars and error bars represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0088] FIGS. 14A-14C shows that optimal reporter plasmid concentration can vary across sensors, in accordance with some embodiments of the present disclosure. FIG. 14A shows absorbance data for T7RNAPNsv-NBl / LaG27-T7RNAPcwith either 0.5 nM, 0.1 nM, or 0.02 nM pT7LacZ reporter plasmid. FIG. 14B shows T7RNAPNev-LaG2 / LaG27-T7RNAPcwith either 0.5 nM, 0.1 nM, or 0.02 nM pT7LacZ reporter plasmid. FIG. 14C shows a heatmap of ABC values for both sensors at all reporter plasmid concentrations. Shaded areas represent the standard deviation of the mean of n=3 technical replicates.

[0089] FIG. 15 shows spontaneous reassembly of T7RNAP fragments with NBs fused to different termini, in accordance with some embodiments of the present disclosure. All T7RNAPNfragments here are wild type to enable spontaneous reassembly. The evolved T7RNAPC,L2A variant enables C-terminal fusions. All reactions contained 0.1 nM pT7LacZ. Symbols represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0090] FIG. 16 shows direct ELISA validation of anti-mCherry NB expression in a CFE system, in accordance with some embodiments of the present disclosure. TTR was used as the control protein. Bars and error bars represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0091] FIGS. 17A-17B show detection range and LOD of the T7RNAPNev-LaM4 / LaM2- T7RNAPCTLISA, in accordance with some embodiments of the present disclosure. FIG. 17A shows absorbance data with increasing concentrations of mCherry or control protein showingan LOD of 50 nM mCherry. FIG. 17B shows absorbance values at 40 minutes showing the detection range. All reactions had 0.05 nM pT7LacZ. TTR was used as the control protein. Bars represent the arithmetic mean ± standard deviation of n=3 technical replicates (white diamonds).

[0092] FIG. 18 shows absorbance data corresponding to the data in FIG. 4B, in accordance with some embodiments of the present disclosure. Columns represent different T7RNAPCNB fusions and row represent different T7RNAPNev NB fusions. Shaded areas represent the standard deviation of the mean of n=3 technical replicates. Consistent with FIG. 4A, the purple curve is for 200 nM mCherry, the yellow curve is for 200 nM control protein (eGFP), and the gray curve is for the buffer-only control.

[0093] FIGS. 19A-19C depict that TLISA can use different protein outputs and different in vitro transcription and translation systems, in accordance with some embodiments of the present disclosure. FIG. 19A shows a sensor circuit for the T7RNAPNev-LaM2 / LaM4- T7RNAPCmCherry TLISA using sfGFP as a protein reporter. Fluorescent measurements (ex. 485 nm, em. 510 nm) after 3 hours of incubation at 37 °C showing detection of mCherry when using (FIG. 19B) an in-house prepared crude lysate-based reaction and when using (FIG. 19C) the commercial myTXTL Sigma 70 kit. Blank represents the background fluorescence of the cell-free reaction and contains everything in the buffer control condition expect pT7sfGFP plasmid. 200 nM TTR was used as the control protein. Bars represent the arithmetic mean ± standard deviation of n=3 technical replicates (white diamonds).

[0094] FIG. 20 shows visualization of the anti-RBD NB and DARPin binding sites, in accordance with some embodiments of the present disclosure. RBD molecule (PDB 6M0J) is shown in gray, and residues that interact with each NB or DARPin are shown in purple. Binding residues for VHHE and VHHV were determined by Koenig et al. (Koenig et al., “Structure-Guided Multivalent Nanobodies Block SARS-CoV-2 Infection and Suppress Mutational Escape,” Science 371 :eabe6230 (2021), which is hereby incorporated by reference in its entirety) and the binding residues for FSR22 were determined by Chonira et al. (Chonira et al., “A Potent and Broad Neutralization of SARS-CoV-2 Variants of Concern by DARPins,” Nat Chem Biol 19:284-291 (2023), which is hereby incorporated by reference in its entirety). Illustrations were made using PyMol.

[0095] FIG. 21 shows T7RNAPNev-VHHV / VHHE-T7RNAPC SARS-CoV-2 RBD TLISA, in accordance with some embodiments of the present disclosure. 500 nM mCherry was usedas the control protein for all experiments. Symbols represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0096] FIGS. 22A-22B show visual detection of SARS-CoV-2 RBD, in accordance with some embodiments of the present disclosure. FIG. 22A shows photos of visible reaction colors at different time points for the T7RNAPNev-FSR22 / VHHV-T7RNAPcSARS-CoV-2 RBD TLISA using 0.1 nM pT7LacZ. Reactions with 500 nM RBD are more red than reactions containing an off-target control protein (mCherry) or just protein buffer after 45 minutes. Detection is still distinguishable at 60 minutes but leaky expression in the off state makes interpretation more difficult. FIG. 22B shows results using 50% lower pT7LacZ (0.05 nM) in this TLISA reaction reduces leak, making reactions appear more visually distinct for a longer period of time. However, doing so also decreases the overall reaction rate.

[0097] FIGS. 23A-23B show TLISA detection of the SARS-CoV-2 S protein, in accordance with some embodiments of the present disclosure. FIG. 23A shows absorbance data of the T7RNAPNSV-FSR22 / VHHV-T7RNAPCshowing detection of 150 nM SARS-CoV-2 S protein. FIG. 23B shows pictures of visible reaction colors after 45 minutes of incubation. Despite background expression in the PBS and off-target protein (MERS CoV-2 S) controls, reactions containing 150 nM SARS-CoV-2 S protein are clearly more red.

[0098] FIGS. 24A-24B depicts determination of the detection range and LOD of the T7RNAPNev-FSR22 / VHHV-T7RNAPcbiosensor for SARS-CoV-2 RBD, in accordance with some embodiments of the present disclosure. FIG. 24A shows absorbance data with increasing concentrations of RBD or control protein showing an LOD of 200 nM RBD. FIG. 24B shows absorbance values after 60 minutes of incubation at 37 °C. All reactions had 0.1 nM pT7LacZ. mCherry was used as the control protein. Bars represent the arithmetic mean ± standard deviation of n=3 technical replicates (white diamonds).

[0099] FIGS. 25A-25E depict results of TTR sensor tuning, in accordance with some embodiments of the present disclosure. FIG. 25A shows genetic sensing circuit for the TTR TLISA. FIG. 25B shows absorbance data for TTR data with either a 7 AA linker (circles) or a 14 AA linker (triangles) on the T7RNAPNSV-NB03 fragment with 0.12 nM pT7LacZ. FIG. 25C shows ABC values for the data in FIG. 25B. FIG. 25D shows absorbance data for the TTR sensor using the 7 AA linker on the T7RNAPNev-NB03 fragment with 0.05 nM pT7LacZ. FIG. 25E shows ABC values for 7 AA linker data in FIGS. 25B and 25D. 500 nM mCherry wasused as the control protein for all experiments. Symbols represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0100] FIGS. 26A-26B show absorbance time course data corresponding to FIGS. 6B and 6C, in accordance with some embodiments of the present disclosure. FIG. 26A shows absorbance data for the T7RNAPNsv-LaM4 / LaM2-T7RNAPcmCherry TLISA in various concentrations of either pooled human saliva or serum as presented in FIG. 6B. FIG. 26B shows absorbance data for the T7RNAPNev-FSR22 / VHHV-T7RNAPcSARS-CoV-2 RBD TLISA with and without pooled human saliva as presented in FIG. 6C. Shaded areas represent the standard deviation of the mean of n=3 technical replicates.

[0101] FIGS. 27A-27B show that naproxen restores vibrancy in colorimetric reactions in serum, in accordance with some embodiments of the present disclosure. FIG. 27A show absorbance values after 45 minutes of incubation showing detection of 200 nM mCherry in 20% pooled human serum with and without added naproxen. Naproxen is known to quench transcription, which explains why final absorbance values are lower when naproxen is added. FIG. 27B shows pictures of reactions after 45 minutes of incubation showing that the addition of naproxen restores vibrancy back to reaction colors. Bars represent the arithmetic mean ± standard deviation of n=3 technical replicates (white diamonds).

[0102] FIG. 28 shows that increasing concentrations of pT7LacZ improves rate of reaction when sensing at room temperature, in accordance with some embodiments of the present disclosure. Absorbance data for T7RNAPNev-FSR22 / VHHV-T7RNAPcSARS-CoV-2 RBD TLISA reaction in 20% v / v pooled human saliva incubated at 25 °C. 200 nM mCherry was used as the control protein. Symbols and error bars represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0103] FIGS. 29A-29F show successful lyophilization of cell-free reactions after 1 hour preexpression reactions, in accordance with some embodiments of the present disclosure. FIG. 29A shows lyophilized reactions after pre-expression of either a plasmid encoding full T7RNAP or an empty reaction (neg. Ctrl) and rehydrated with 0.05 nM pT7LacZ. FIG. 29B shows lyophilized reactions after pre-expression of plasmids encoding WT split T7RNAP fragments or an empty reaction (neg. Ctrl) and rehydrated with 0.05 nM pT7LacZ. FIG. 29C shows lyophilized T7RNAPNev-LaM4 / LaM2-T7RNAPC mCherry TLISA rehydrated with 0.1 nM pT7LacZ. FIG. 29D shows lyophilized T7RNAPNev-FSR22 / VHHV-T7RNAPC SARS-CoV-2 RBD TLISA rehydrated with 0.5 nM pT7LacZ. FIG. 29E shows that lyophilizedT7RNAPNev-FSR22 / VHHV-T7RNAPC SARS-CoV-2 RBD TLISA rehydrated with 0.5% v / v RNAse Inhibitor and 0.75 nM pT7LacZ and 0% v / v human saliva or FIG. 29F 20% v / v human saliva, corresponding to FIG. 6D. Symbols represent the arithmetic mean ± standard deviation of n=3 technical replicates.

[0104] FIGS. 30A-30C show leak assessment of the split T7RNAP in a CFE system, in accordance with some embodiments of the present disclosure. FIG. 30A shows the wt T7RNAPNfragment (left), the T7RNAPNSVfragment (middle), and the T7RNAPCfragment (right) alone have negligible activity at 20 nM and 10 nM of plasmid. FIG. 30B shows relative to the wt T7RNAPNfragment, T7RNAP Nev has high levels of spontaneous reassembly with T7RNAPC. FIG. 30C shows that in the absence of antigen, the T7RNAPNSV and T7RNAPCfragments with different NB fusions have some spontaneous reassembly. Lower concentrations of T7RNAP fragments and pT7LacZ result in less LacZ activity. Shaded areas represent the standard deviation of the mean of n=3 technical replicates.

[0105] FIG. 31 shows SDS-PAGE analysis of purified proteins used in this study, in accordance with some embodiments of the present disclosure. The additional bands and high apparent MW of mCherry (Lanes F30 and F31) are expected and not evidence of impurities; the bands appearing at 20 kDa and 10 kDa are a result of mCherry fragmentation due to denaturing by boiling. Cong et al., “Schellenberg, High-Efficiency Recombinant Protein Purification Using mCherry and YFP Nanobody Affinity Matrices,” Protein Science 31 :e4383 (2022) and Gross, et al., “The Structure of the Chromophore within DsRed, a Red Fluorescent Protein from Coral,” Proceedings of the National Academy of Sciences 97:11990-11995 (2000), both of which are hereby incorporated by reference in their entirety).

[0106] FIGS. 32A-32B illustrate the use of cell-surface TLISA to detect native Globo-H tumor associated carbohydrate antigen. FIG. 32A shows a depiction of cell-surface TLISA. T7RNAP fragments each translationally fused to the GH46 anti-Globo-H nanobody (NB) (Khilji et al., “Generation of Glycan-Specific Nanobodies,” Cell Chemical Biology 29: 1353-1361 ,e6 (2022), which is hereby incorporated by reference in its entirety) are pre-expressed in a cell-free reaction as previously described. Cells cultured in clear, flat-bottomed tissue culture treated polystyrene 384-well plates are washed once with PBS to remove culture media. 15 uL of the pre-expressed TLISA reaction is then added to each well with pT7LacZ reporter plasmid and CPRG substrate and incubated at 37°C. Only if Globo-H TACAs are present will the T7RNAP fragment colocalize and drive the expression of LacZ reporter. FIG. 32A depicts absorbancevalues after 22 minutes of incubation showing TLISA detection of Globo-H on A549 cells relative to the Globo-H negative control HEK293T cells. Higher cell densities lead to a more distinct TLISA response. Plotted are the mean and standard deviation of n=3 technical replications (white diamonds). Asterisks indicate a significant difference between two samples as determined by the results of a two-tailed t test (****p < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05).DETAILED DESCRIPTION

[0107] To facilitate an understanding of the principles and features of the present disclosure, various illustrative embodiments are explained below. The components, steps, and materials described hereinafter as making up various elements of the embodiments disclosed herein are intended to be illustrative and not restrictive. Many suitable components, steps, and materials that would perform the same or similar functions as the components, steps, and materials described herein are intended to be embraced within the scope of the disclosure. Such other components, steps, and materials not described herein can include, but are not limited to, similar components or steps that are developed after development of the embodiments disclosed herein.

[0108] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0109] Also, in describing the preferred exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

[0110] Ranges can be expressed herein as from “about” or “approximately” one particular value and / or to “about” or “approximately” another particular value. When such a range is expressed, another exemplary embodiment includes from the one particular value and / or to the other particular value.

[0111] By ‘ ‘comprising” or “containing” or “including” is meant that at least the named compound, member, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, evenif the other such compounds, material, particles, method steps have the same function as what is named.

[0112] Mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0113] The materials described as making up the various members of the invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, for example, materials that are developed after the time of the development of the invention.

[0114] The sequences disclosed herein may include wild type, variants, mutants, and derivatives thereof. As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As described herein, a “variant, “mutant,” or “derivative” may include a molecule having a sequence that differs from a reference molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. A variant or mutant may include a fragment of a reference molecule. For example, a mutant or variant molecule may one or more insertions, deletions, or substitution of at least one amino acid residue relative to a reference molecule. Unless indicated to the contrary, sequences having less than 100% similarity with disclosed sequences may be used in the methods, kits, and systems of the present disclosure.

[0115] Regarding proteins, percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequencesshown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0116] A first aspect of the present disclosure provides a method for detecting an analyte level in a sample. The method includes providing a first RNA polymerase fragment comprising a first analyte affinity domain bound thereto at an end of the first RNA polymerase fragment, the first analyte affinity domain configured to bind to a target analyte; providing a second RNA polymerase fragment comprising a second analyte affinity domain bound thereto at an end of the second RNA polymerase fragment, the second analyte affinity domain configured to bind to a target analyte; providing a biological sample comprising a target analyte; adding the biological sample comprising the target analyte to the first RNA polymerase fragment and second RNA polymerase fragment to form a reaction mixture; and subjecting the reaction mixture to a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output.

[0117] The terms “target,” “target sequence”, “target region”, and “target nucleic acid,” as used herein, are used interchangeably and are synonymous. The terms refer to a region or sequence of an analyte which is to be detected. A target analyte or target as described herein may include clinically relevant analytes, i.e. analytes, that may be used for determining whether or not a subject, such as a human being, has a condition, such as a disease. The target analyte or target described herein may include a portion of sequence in the sample which is to be detected or analyzed. The term target includes all variants of the target sequence, e.g., one or more mutant variants and, or a wild-type variant. In one embodiment, the analyte may be a protein, a glycan, a small molecule, a virus particle, or any combination thereof. In one embodiment, the analyte is a protein. The analyte may, in some embodiments, be a carbohydrate. Alternatively, the analyte may, in some embodiments, be a virus particle.

[0118] Turning to the target analyte (i.e, analyte-of-interest), the methods described here can be used to detect any analyte-of-interest (i.e., target analyte), including small molecule, peptide, or polypeptide analytes. In some embodiments, the analyte comprises at least one of a small molecule, a protein, a glycan, a virus particle, a peptide, a polypeptide, a protein, a glycoprotein, and the like. In some embodiments, the analyte is from a pathogenic organism selected from the group consisting of bacteria, viruses, protozoa, worms, fungi, and the like. In one embodiment, the analyte comprises RNA from a virus. In some embodiments, the virus is a SARS-CoV-2.

[0119] As described herein, the terms “protein,” “peptide,” and “polypeptide,” refer to molecules comprising a chain a polymer of amino acid residues joined by amide linkages. The term “amino acid residue,” includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include nonstandard or unnatural amino acids. The term “amino acid residue” may include alpha-, beta-, gamma-, and delta-amino acids.

[0120] As used herein, a “peptide” may be a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2ndedition, Brooks & Cole 110 (1999), which is hereby incorporated by reference in its entirety). In some embodiments, a peptide as contemplated herein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. A polypeptide, also referred to as a protein, may be a length >100 amino acids (Garrett & Grisham, Biochemistry, 2ndedition, Brooks & Cole 110 (1999), which is hereby incorporated by reference in its entirety). A polypeptide, as contemplated herein, may comprise, but is not limited to, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues.

[0121] The proteins disclosed herein may include “wild type” proteins and variants, mutants, and derivatives thereof. As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As described herein, a “variant, “mutant,” or “derivative” may include a protein molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have oneor more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. A variant or mutant may include a fragment of a reference molecule. For example, a mutant or variant molecule may one or more insertions, deletions, or substitution of at least one amino acid residue relative to a reference polypeptide.

[0122] Regarding proteins, the phrases “percent identity” and “% identity,” refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0123] Regarding proteins, percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0124] The disclosed proteins may be substantially isolated or purified. The term “substantially isolated or purified” refers to proteins that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0125] Exemplary protein antigens that may be used in accordance with the present disclosure include but are not limited to those described in Table 2, infra, eGFP (e.g., SEQ ID NO: 78), mCherry (e.g., SEQ ID NO: 79), SARS-CoV2 RBD (e.g., SEQ ID NO: 80), Transthyretin (TTR) (e.g., SEQ ID NO: 81), SARS-CoV-2 Hexapro S ectodomain (e.g., SEQ ID NO: 82) (see Hsieh et al., “Structure-Based Design of Prefusion-Stabilized SARS-CoV-2 Spikes,” Science 369:1501-1505 (2020), which is hereby incorporated by reference in its entirety), and MERS-CoV 2P S ectodomain ) (e.g., SEQ ID NO: 81) (see Pallesen et al., “Immunogenicity and Structures of a Rationally Designed Profusion MERS-CoV Spike Antigen,” Proceedings of the National Academy of Sciences 114:E7348-E7357 (2017), which is hereby incorporated by reference in its entirety).

[0126] The methods described herein may, in various embodiments, be conducted in a cell- free environment. As used herein, “cell-free environment” or “cell-free system” refers to a set of reagents (e.g., RNA polymerases and analyte affinity domains) capable of providing for or supporting a reaction in vitro or ex vivo. In any of the embodiments disclosed herein, the first RNA polymerase fragment, second RNA polymerase fragment, and biological sample may be added into a cell-free expression mixture, the mixture comprising lysates of cells capable of executing transcription and translation. The disclosed cell-free environment may utilize components that are crude and / or that are at least partially isolated and / or purified. As used herein, the term “crude” may mean components obtained by disrupting and lysing cells and optionally purifying the crude components from the disrupted and lysed cells, for example by centrifuging the disrupted and lysed cells and collecting the crude components from the supernatant and / or pellet after centrifugation. The term “isolated or purified” may include components that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0127] An analyte affinity domain as described herein refers to both a first analyte affinity domain and a second analyte affinity domain. In any of the embodiments disclosed herein, the first analyte affinity domain may be a protein affinity domain, or the second analyte affinity domain is a protein affinity domain, or both the first and second analyte affinity domains are protein affinity domains. As described in the embodiments disclosed herein, the first analyte affinity domain may fuse to a first RNA polymerase fragment and a second analyte affinity domain fuses to a second RNA polymerase fragment in the presence of an antigen. In thepresence of the antigen, both analyte affinity domains bind and bring both RNA polymerase fragments in close proximity, driving reassembly. The reassembled RNA polymerase then drives expression of the target analyte. The target analyte may, for example, generate a range of visually interpretable output colors.

[0128] In any of the embodiments disclosed herein, the first analyte affinity domain may be, for example, a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof. In one embodiment, the first analyte affinity domain comprises a nanobody. In another embodiment, the first analyte affinity domain comprises a monobody. In yet another embodiment, the first analyte affinity domain comprises a DARPin.

[0129] In any of the embodiments disclosed herein, the second analyte affinity domain may be, for example, a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof. In one embodiment, the second analyte affinity domain comprises a nanobody. In another embodiment, the second analyte affinity domain comprises a monobody. In yet another embodiment, the second analyte affinity domain comprises a DARPin.

[0130] Examples of analyte affinity domains which may be used in accordance with the present disclosure are shown in Table 5. Exemplary nanobodies that may be used in the present disclosure include but are not limited to NB01 (SEQ ID NO: 88), LaG2 (SEQ ID NO: 89), LaG14 (SEQ ID NO: 90), LaG19 (SEQ ID NO: 91), LaG26 (SEQ ID NO: 92), LaG27 (SEQ ID NO: 93), all of which have a target antigen of eGFP. Additional exemplary nanobodies that may be used in the present disclosure include but are not limited to LaM2 (SEQ ID NO: 96), LaM3 (SEQ ID NO: 97), LaM4 (SEQ ID NO: 98), and LaM6 (SEQ ID NO: 99), all of which have a target antigen of mCherry. Further nanobodies that may be used in accordance with the present disclosure include VHHE (SEQ ID NO: 100) and VHHV (SEQ ID NO: 101), which have a target antigen of SARS CoV-2 RBD, and NB03 (SEQ ID N: 103), which has a target antigen of TTR. A suitable monobody that may be used in accordance with the present disclosure includes but is not limited to GS2 (SEQ ID NO: 94) which has a target antigen of eGFP. Suitable DARPins which may be used in accordance with the present disclosure include but are not limited to 3G86.32 (SEQ ID NO: 95) which has a target antigen of eGFP, and FSR22 (SEQ ID NO: 102) which has a target antigen of SARS CoV-2 RBD. In any of theembodiments disclosed herein, the first analyte affinity domain may be, for example, NB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32. In any of the embodiments disclosed herein, the second analyte affinity domain maybe, for example, NBO3, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

[0131] In any of the embodiments disclosed herein, the first analyte affinity domain and the second analyte affinity domain may include minimally overlapping epitopes configured to allow the first analyte affinity domain and the second analyte affinity domain to bind simultaneously to the target analyte.

[0132] As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “RNA polymerase” catalyzes the chemical reactions that synthesize RNA from a DNA template. Known RNA polymerases include, for example, RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V. Examples include, but are not limited to T3 RNA polymerase, T7 RNA polymerase, Hi-T7® RNA Polymerase, SP6 RNA Polymerase, E. coli Poly(A) Polymerase, Poly(U) Polymerase, E. coli RNA Polymerase, Core Enzyme, and E. coli RNA Polymerase, Holoenzyme, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.

[0133] In any of the embodiments disclosed herein, the first RNA polymerase fragment and the second RNA polymerase fragment may be configured to have minimal spontaneous reassembly. In any of the embodiments disclosed herein, the first RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase. In any of the embodiments disclosed herein, the first RNA polymerase fragment comprises a split T7 RNA polymerase. In any of the embodiments disclosed herein, the first RNA polymerase fragment comprises T7RNAPN(e.g, SEQ ID NO: 84), T7RNAPnev (e.g., SEQ ID NO: 85), T7RNAPC(e.g., SEQ ID NO 86), or T7RNAPC.L2A (e.g., SEQ ID NO: 87). In any of the embodiments disclosed herein, the second RNA polymerase fragment is derived from a singlesubunit RNA polymerase or a multi-subunit RNA polymerase. In any of the embodiments disclosed herein, the second RNA polymerase fragment comprises a split T7 RNA polymerase. In any of the embodiments disclosed herein, the second RNA polymerase fragment comprisesT7RNAPN(e.g., SEQ ID NO: 84), T7RNAPnev (e.g., SEQ ID NO: 85), T7RNAPC(e.g., SEQ ID NO 86), or T7RNAPC,L2A (e.g., SEQ ID NO: 87). Exemplary sequences of T7RNAP fragments that may be used in accordance with the present disclosure include, but are not limited to, those shown in Table 3.

[0134] In any of the embodiments disclosed herein, when the first RNA polymerase fragment and the second RNA polymerase fragment are forcibly colocalized upon binding of the first and second affinity domains to the target analyte, this drives a reassembly of the first RNA polymerase fragment and the second RNA polymerase fragment, turning on a reporter gene, and generating the measurable output.

[0135] A “sample,” “biological sample,” “test sample,” “specimen,” “sample from a subject,” and “patient sample” as used herein may be used interchangeably and may be a sample of blood, such as whole blood, tissue, skin, urine, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample may be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0136] Suitable biological samples in accordance with the present disclosure include biological samples such as blood, blood serum, blood plasma, cerebrospinal fluid, urine, saliva, tissue. In any of the embodiments disclosed herein, the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, mucus, lymph, interstitial fluid, cerebrospinal fluid, pus, breast milk, and amniotic fluid.

[0137] The phrase “derived from” as used herein includes cells or a biological sample and indicates that the cells or the biological sample were obtained from the stated source at some point in time. For example, a cell derived from a subject can include a blood cell obtained directly from the subject (e.g., unmodified). In some instances, a cell derived from a given source undergoes one or more rounds of cell division and / or cell differentiation such that the original cell no longer exists, but the continuing cell (e.g., daughter cells from all generations) will be understood to be derived from the same source. The term includes directly obtained from, isolated and cultured, or obtained, frozen, and thawed. The term “derived from” may also refer to a component or fragment of a cell obtained from a tissue or cell, including, but not limited to, a protein, a nucleic acid, a membrane or fragment of a membrane, and the like.

[0138] The term “isolating” or “isolated” as described herein, when referring to a cell or a molecule (e.g., nucleic acids or protein), includes where the cell or molecule is or has been separated from its natural, original or previous environment. For example, an isolated cell can be removed from a tissue derived from its host individual, but can exist in the presence of other cells (e.g., in culture).

[0139] A Limit of Detection (LoD) as described herein may include the lowest concentration of the measured component (i.e. a quantity intended to be measured) that can be detected at a specified level of confidence. The level of confidence is typically 95%, with a 5% likelihood of a false negative measurement. LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. LoD may be determined by utilizing both the measured LoB and test replicates of a sample known to contain a low concentration of analyte. A Limit of Quantitation (LoQ) as described herein refers to the lowest concentration at which the analyte can be reliably detected and also at which some predefined goals for bias and imprecision are met. An LoQ may be equivalent to an LoD or it could be at a much higher concentration.

[0140] The term “reaction mixture,” as used herein, refers to a solution containing components necessary to carry out a given reaction or to create conditions under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output. A reaction mixture is referred to as complete if it contains all reagents or components necessary to perform the reaction (i.e., create a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output). Components for a reaction mixture may be stored separately in separate container, each containing one or more of the total components. Components may be packaged separately for commercialization and useful commercial kits may contain one or more of the reaction components for a reaction mixture.

[0141] In any of the embodiments disclosed herein, the measurable output comprises absorbance, fluorescence, luminescence, or a combination thereof.

[0142] In any of the embodiments disclosed herein, the measurable output comprises absorbance, fluorescence, or luminescence at 580 nm.

[0143] In some embodiments, the method includes quantifying the signal and determining a concentration of the analyte in the biological sample. In some embodiments, the signal is a fluorescent signal, a bioluminescent signal, a chemical signal, an electrochemical signal, or acolorimetric signal. In some embodiments, the analyte is present in the sample at a concentration ranging from about 1 IM to about 500 LIM or about 1 mM. In some embodiments, the analyte is present in the sample at a concentration ranging from about 500 fM to about 500 pM or about 1 mM. In some embodiments, the analyte is present in the sample at a concentration ranging from about 1 pM to about 500 pM. In some embodiments, the analyte is present in the sample at a concentration ranging from about 500 pM to about 500 pM. In some embodiments, the analyte is present in the sample at a concentration ranging from about 1 nM to about 500 pM.

[0144] In any of the embodiments disclosed herein, the method further includes providing a linker sequence in the first RNA polymerase fragment, the second RNA polymerase fragment, or in both the first and second RNA polymerase fragments. In any of the embodiments disclosed herein, the linker sequence is between 5 and 30 amino acids in length. For example, the linker may be about 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, or 30 amino acids in length. Example linkers that may be used in accordance with the embodiments described herein include Linkerl4 (e.g., SEQ ID NO: 104), Linker7 (e.g., SEQ ID NO: 105), and Linker28 (e.g., SEQ ID NO: 106). In any of the embodiments disclosed herein, the linker sequence comprises the amino acid sequences SEQ ID NOS: 104- 106.

[0145] In accordance with any of the embodiments disclosed herein, the first RNA polymerase fragment may, for example, be lyophilized, the second RNA polymerase fragment may, for example, be lyophilized, or both the first RNA polymerase fragment and the second RNA polymerase fragment may, for example, be lyophilized. Lyophilization in accordance with the present disclosure includes methods in accordance with conventional techniques, which are known to those skilled in the art. Lyophilization is also referred to herein as freeze-drying. Lyophilizable formulations can be reconstituted into solutions, suspensions, emulsions, or any other suitable form for administration or use. Lyophilizable formulations are typically first prepared as liquids, then frozen and lyophilized. The total liquid volume before lyophilization can be less than, equal to, or more than, the final reconstituted volume of the lyophilized formulation. The lyophilization process is known to those of ordinary skill in the art, and typically includes sublimation of water from a frozen formulation under controlled conditions.

[0146] Lyophilized formulations in accordance with the present disclosure can be stored at a wide range of temperatures. Lyophilized formulations may be stored below 25° C., forexample, refrigerated at 2-8° C., or at room temperature (e.g., approximately 25° C.). Stability of a lyophilized formulation may be determined in a number of ways known in the art, for example, by visual appearance and / or cake and / or moisture content.

[0147] Freeze-dried or lyophilized formulations are typically prepared from liquids, that is, from solutions, suspensions, emulsions, and the like. Thus, the liquid that is to undergo freeze- drying or lyophilization preferably comprises all components desired in a final reconstituted liquid formulation. As a result, when rehydrated or reconstituted, the freeze-dried or lyophilized formulation will render a desired liquid formulation upon reconstitution.

[0148] As shown in FIG. 1A, in accordance with the present disclosure, the first RNA polymerase fragment (e.g., T7RNAPNCV) can fuse to a first analyte affinity domain (e.g., a nanobody) and the second RNA polymerase fragment (e.g., T7RNAPC) can fuse to a separate, second, analyte affinity domain (e.g., a nanobody) in the presence of an antigen. In the presence of antigen, both analyte affinity domains can bind and bring both RNA polymerase fragments in close proximity, driving reassembly. The reassembled RNA polymerase can then drive expression of the target analyte. The target analyte may generate a range of visually interpretable output colors.

[0149] The steps of the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The steps may be repeated or reiterated any number of times to achieve a desired goal unless otherwise indicated herein or otherwise clearly contradicted by context.

[0150] Another aspect of the present disclosure provides a kit for detecting an analyte level in a sample. The kit includes a first RNA polymerase fragment comprising a first analyte affinity domain bound thereto at an end of the first RNA polymerase fragment, the first analyte affinity domain configured to bind to a target analyte; and a second RNA polymerase fragment comprising a second analyte affinity domain bound thereto at an end of the second RNA polymerase fragment, the second analyte affinity domain configured to bind to a target analyte.

[0151] Such kits contain monitors, reagents and procedures that can be utilized in a clinical or research setting or adapted for either the field laboratory or on-site use. In particular, kits comprising the disclosed reagents used in practicing the methods described herein include any of a number of means for detecting the target analyte and measuring the output produced subsequent to capture, along with appropriate instructions, are contemplated Suitable kits comprise reagents sufficient for performing an assay to detect a target analyte.

[0152] It is to be understood that such a kit is useful for any of the methods of the present invention. The choice of particular components is dependent upon the particular method the kit is designed to carry out. Additional components can be provided for detection of the analytical output, as measured by detection of a measurable output.

[0153] As described above, the kit optionally further comprises instructions for detecting the target analyte by the methods described herein. The instructions present in such a kit instruct the user on how to use the components of the kit to perform the various methods of the present application. These instructions can include a description of the detection methods of the invention, including detection by measuring output.

[0154] In some examples, one or more of the methods described herein are performed in a vessel, e.g., a single, vessel. The term “vessel,” as used herein, refers to any container suitable for holding on or more of the reactants (i.e. components) described herein. Examples of vessels include, but are not limited to, a microtitre plate, a test tube, a microfuge tube, a beaker, a flask, a multi-well plate, a cuvette, a flow system, a microfiber, a microscope slide and the like.

[0155] This aspect includes various embodiments in accordance with the previously described aspect. The described target analyte, analyte affinity domains, RNA polymerase fragments, biological sample in the previously described aspects are likewise applicable in to the kit described in this aspect.

[0156] In any of the embodiments disclosed herein, the first RNA polymerase fragment and the second RNA polymerase fragment may be mixed with a biological sample to form a reaction mixture, in accordance with the previously described aspect.

[0157] In any of the embodiments disclosed herein, the reaction mixture is subject to a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output, in accordance with the previously described aspect.

[0158] In any of the embodiments disclosed herein, the first RNA polymerase fragment and the second RNA polymerase fragment are configured to have minimal spontaneous reassembly when subject to said condition, in accordance with the previously described aspect.

[0159] In any of the embodiments disclosed herein, when the first RNA polymerase fragment and the second RNA polymerase fragment are forcibly colocalized upon binding of the first and second affinity domains to the target analyte, thus driving a reassembly of the first RNApolymerase fragment and the second RNA polymerase fragment, turning on a reporter gene, and generating the measurable output, in accordance with the previously described aspect.

[0160] In any of the embodiments disclosed herein, the first fragment, second fragment, and biological sample are added into a cell-free expression mixture, the mixture comprising lysates of cells capable of executing transcription and translation, in accordance with the previously described aspect.

[0161] In accordance with the embodiments described herein, the present disclosure may, other aspects, relate to platforms, systems, and devices that may include substantially the same components as described herein for performing the methods described herein.

[0162] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

[0163] Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.

[0164] Furthermore, the purpose of the foregoing Abstract is to enable the United States Patent and Trademark Office and the public generally, and especially including the practitioners in the art who are not familiar with patent and legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the claims of the application, nor is it intended to be limiting to the scope of the claims in any way.EXAMPLES

[0165] The following Examples are presented to illustrate various aspects of the present application, but are not intended to limit the scope of the claimed application.Materials and Methods

[0166] Bacterial Strains and Plasmid Preparation - DNA oligonucleotides for cloning and sequencing were synthesized by Eurofins Genomics. T7RNAP fragments were amplified via PCR from the BL21 (DE3) genome. Gene strands for T7RNAPN„, and all nanobody, monobody, and DARPin sequences were codon optimized and synthesized by Twist Biosciences. Plasmids were cloned by either Gibson Assembly (Gibson et al., “Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases,” Nat Methods 6:343-345 (2009), which is hereby incorporated by reference in its entirety) or blunt-ended ligation using the pJLl plasmid backbone. E. coli strains DHIOp and DH5a were used for cloning and plasmid preparations. Isolated colonies were grown overnight in LB medium with either kanamycin sulfate (33 pg / mL) or tetracycline (15 pg / mL). Plasmid DNA from overnight cultures was purified using EZNA mini prep columns (OMEGA Bio-Tek). Plasmid sequences were verified with Sanger DNA sequencing (Eurofins Genomics). Sequence-confirmed plasmids were then purified using EZNA midiprep or maxiprep columns (OMEGA Bio-Tek), followed by isopropanol and ethanol precipitation. The purified DNA pellet was reconstituted in elution buffer, measured on a Nanodrop 2000 for concentration, and stored at -20 °C until use. E. coli strain BL21 AlacIZYA was created by lambda red recombination (Datsenko, and B. L. Wanner, “One-Step Inactivation of Chromosomal Genes in Escherichia coli K-12 Using PCR Products,” Proceedings of the National Academy of Sciences 97:6640-6645 (2000), which is hereby incorporated by reference in its entirety) and used for in-house cell-free lysate preparation.

[0167] A list of exemplary PCT primers that may be used for plasmid cloning in accordance with the present disclosure is shown in Table 1 .Table 1. PCR primers used for plasmid cloning.

[0168] Preparation of cell-free lysate - Bacterial lysate for all experiments was prepared as described by Kwon and Jewett (Y.-C. Kwon and M. C. Jewett, “High-Throughput Preparation Methods of Crude Extract for Robust Cell-Free Protein Synthesis,” Scientific Reports 5:8663 (2015), which is hereby incorporated by reference in its entirety) with a few protocol modifications. BL21 AlacIZYA cells were grown in 2xYTP medium at 37 °C and 220 rprn to an optical density (OD) between 1.5 -2.0, corresponding to the mid-exponential growth phase. Cells were centrifuged at 4 °C and 2700 x g and washed via resuspension with S30 buffer (10 mM tris-acetate [pH 8.2], 14 mM magnesium acetate, 60 mM potassium acetate, and 2 mM dithiothreitol). These centrifugation and wash steps were repeated twice for a total of three S30 washes. After the final centrifugation, the wet cell mass was measured, and cells were resuspended in 1 mL S30 buffer per 1 g of wet cell mass. The cellular resuspension was divided into 0.5 mL aliquots. Cells were lysed using a Q125 sonicator (Qsonica) at a frequency of 20 kHz and 50% amplitude. Cells were sonicated on ice with cycles of 10 seconds on and 10 seconds off, delivering approximately 150 J, at which point the cells appeared visibly lysed. An additional 4 mM dithiothreitol was added to each tube immediately after lysing, and the sonicated mixture was then centrifuged at 12,000 x g and 4 °C for 10 minutes. Aftercentrifugation, the supernatant was divided into 1 mL aliquots, and incubated at 37 °C and 220 rpm for 80 minutes. After this runoff reaction, the lysate was centrifuged at 12,000 x g and 4 °C for 10 minutes. The supernatant was removed and loaded into a 10 kDa molecular weight cutoff dialysis cassette (Thermo Fisher). Lysate was dialyzed in 1 L of S30B buffer (14 mM magnesium glutamate, 60 mM potassium glutamate, 1 mM dithiothreitol, and pH-corrected to 8.2 with tris) at 4 °C for 3 hours. Dialyzed lysate was removed and centrifuged at 12,000 x g and 4 °C for 10 minutes. The supernatant was removed, aliquoted, and stored at -80 °C until use.

[0169] TLISA Cell-free Reactions - All TLIS A reactions consisted of a pre-expression reaction and a final sensing reaction. For the pre-expression, cell-free reactions were assembled in 1.5 mL centrifuge tubes as previously described by Kwon and Jewett (Y.-C. Kwon and M. C. Jewett, “High-Throughput Preparation Methods of Crude Extract for Robust Cell-Free Protein Synthesis,” Scientific Reports 5:8663 (2015), which is hereby incorporated by reference in its entirety) with 10 mM arabinose and equimolar concentrations (25 nM) of T7RNAPNev-NB and NB-T7RNAPC plasmids. Reactions were pre-expressed at 37 °C for one hour. Then, pT7LacZ, 0.6 mg / mL CPRG, and purified protein were added to each tube for the final sensing reaction. Each cell-free reaction had a final volume of 10 pL and was pipetted into a clear- bottomed 384-well plate (Greiner Bio-One). The final sensing reaction was carried out in the BioTek Synergy H4 plate reader at 37 °C for one hour with absorbance measurements at 580 nm taken every minute. Plates were sealed with a transparent adhesive film to prevent evaporation. For reactions containing biological samples, the final sensing reaction contained either 10% or 20% pooled human serum (MP Biomedicals) or 10% or 20% pooled human saliva (Innovative Research Inc.) and RNAse inhibitor, murine (New England BioLabs). For reactions using naproxen, the protocol was slightly modified; CPRG was not added to the final reaction. Rather, 40 mM naproxen and 0.6 mg / mL CPRG were added to the final sensing reaction after 45 minutes, and then incubated for another 45 minutes before taking the final measurement.

[0170] Calculating Area Between Curves (ABC) -ABC is defined as a quantitative metric to compare the quality of different TLISA biosensors. ABC is calculated by determining the area between the absorbance curve of the sensor when the target antigen is present and the absorbance curve of the sensor when the control protein is added from 0 to 60 minutes (Equation 1), in some ways analogous to the use of the area under the curve for a receiveroperating characteristic (AUC ROC) to assess performance of diagnostic tests. Hajian-Tilaki, “Receiver Operating Characteristic (ROC) Curve Analysis for Medical Diagnostic Test Evaluation,” Caspian J Intern Med 4:627-635 (2013), which is hereby incorporated by reference in its entirety.EquationIn general, the greater the ABC, the better the sensor quality. Greater ABC indicates a greater difference in visual sensor color over time between on and off conditions. Visual limits of detection at a given time point are determined by the difference in absorbance values (FIG. 8).

[0171] Protein Purification - For the expression and purification of SARS-CoV-2 RBD, SARS-CoV-2 S protein, andMERS-CoV S protein (sequences in Table 2), plasmids containing 6xHis-tagged proteins were transfected into Expi293F suspension cells with the ExpiFectamine 293 transfection kit (Gibco) according to the manufacturer’s protocol.Table 2. Sequences of the protein antigens used in this study.*Hsieh et al., “Structure-Based Design of Prefusion-Stabilized SARS-CoV-2 Spikes,” Science 369:1501-1505 (2020), which is hereby incorporated by reference in its entirety.** Pallesen et al., “Immunogenicity and Structures of a Rationally Designed Prefusion MERS- CoV Spike Antigen,” Proceedings of the National Academy of Sciences 114:E7348-E7357 (2017), which is hereby incorporated by reference in its entirety.Plasmids were codon optimized for expression in mammalian cells and synthesized by Gene Universal Inc. (Newark, DE). Five days after transfection, cultures were centrifuged for 5 minutes at 5000 x g, and the supernatant was thoroughly dialyzed into PBS. Ni-NTA resin was equilibrated with 10 column volumes (CVs) of IMAC binding buffer (150 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 8.0), then dialyzed supernatant was added to the resin. The resin was washed with 20 CVs of binding buffer, and the protein was eluted with 10 CVs of elution buffer (150 mM Tris, 150 mM NaCl, 400 mM imidazole, pH 8.0). Eluted protein was concentrated in a 10 kDa MWCO Amicon spin filter (EMD Millipore) to < 1 mL. Concentrated protein was injected onto a Superdex Increase 200 10 / 300 GL (Cytiva) size exclusion column to remove any remaining impurities. The SARS-CoV-2 RBD was buffer exchanged into S30B buffer and the two S proteins were buffer exchanged into PBS. Concentration was measured by bicinchoninic acid (BCA) assay, and purity was assessed by SDS-PAGE (FIG. 31).

[0172] For the expression and purification of eGFP, mCherry, and TTR, plasmids expressing 6xHis-tagged proteins under pBAD regulation were transformed into BL21 AlacIZYA cells. Single colonies were then grown overnight in 50 mL of 2xYTP medium and 15 pg / mLtetracycline at 37 °C and 220 rpm. The next day, 5 mL of the overnight culture was diluted in 500 mL 2xYTP medium with tetracycline and incubated at 37 °C and 220 rpm in a 1 L baffled flask. Once the OD reached 0.4-0.6, 2 mM arabinose was added to the culture to induce protein expression. Flasks were then transferred to a separate incubator and grown overnight at 25 °C and 180 rpm. The next day, cultures were transferred to 50 mL tubes and centrifuged at 2700 x g and 4 °C for 15 minutes. Cells were washed in IxPBS and centrifuged again under the same conditions. The supernatant was discarded, and cells were resuspended in 2 mL of lysis buffer (50 mM disodium phosphate, 500 mM sodium chloride, 10 mM imidazole, pH 8) per 1 g of wet cell mass. Cells were lysed via sonication using the conditions specified for cell-free lysate preparation except without the addition of dithiothreitol. Lysed cells were centrifuged at 12,000 x g and 4 °C for 15 minutes. Lysates were loaded onto a pre-equilibrated Ni-NTA column and washed with 20 CVs of binding buffer. Each protein was eluted with 10 CVs of elution buffer, then concentrated by spin filter to < 1 mL. Proteins were further purified by size exclusion chromatography. Fractions containing high concentrations of protein were collected and combined. Proteins were buffer exchanged into S30B buffer. Protein purity was assessed via SDS-PAGE (FIG. S25). Protein concentration was determined by absorbance at 280 nm.

[0173] Lyophilization - TLISA pre-expression reactions were prepared as previously described in PCR tubes. After one hour of pre-expression, tubes were transferred to a prechilled Labconco Fast-freeze flask and stored at -80 °C until frozen. The flask was then connected to a Labconco FreeZone benchtop freeze dryer and samples lyophilized at -50 °C and 0.02 mbar for 3 hours. Samples were then removed and rehydrated on ice for the final reaction.

[0174] ELISAs - NB_LacZ fusion proteins were expressed in cell-free reactions. Cell-free reactions were assembled in 1.5 mL centrifuge tubes as previously described by Kwon and Jewett (Kwon and Jewett, “High-Throughput Preparation Methods of Crude Extract for Robust Cell-Free Protein Synthesis,” Scientific Reports 5:8663 (2015), which is hereby incorporated by reference in its entirety) with 5 nM of the NB-LacZ fusion plasmid. Reactions were incubated at 30 °C overnight. Then, cell-free reactions were centrifuged at 12,000 x g and 4 °C for 15 minutes. The supernatant was diluted 1 :4 in lx PBS. 100 uL of 1 mg / mL of purified 6xhis-tagged proteins were added to a Ni-coated 96-well plate and incubated for 2 hours at room temperature on a rocker. Wells were washed four times with wash buffer (IxPBS with 0.05% Tween-20) to remove unbound protein. 100 uL of the diluted NB_LacZ was added to each well and incubated for two hours at room temperature on a rocker. Wells were washedfour times with wash buffer to remove unbound NBs. 100 uL of CPRG was added to each well. The plate was incubated for a final time at 37 °C without shaking. End absorbance was measured using the BioTek Synergy H4 plate reader.

[0175] Color images and processing - Pictures of colorimetric reactions were taken with a Sony Cybershot DSC-WX9. A 3D-printed dome with an aperture at the top was placed over samples for light control. Pictures represent 10 pL reactions in clear, flat-bottomed 384-well plates on top of a white piece of paper. The centers of the wells of interest were cropped using Adobe Illustrator and combined to make color arrays. A brightness filter was uniformly applied to photos to make them better resemble actual appearance.Example 1 - Proof of Principle eGFP TLISA

[0176] As a proof-of-principle demonstration, the first aim was to engineer a TLISA biosensor to detect eGFP as a model protein. The TLISA platform was implemented on three plasmids (FIG. IB): one encoding the evolved N-terminal portion (residues 1-179) of T7RNAP (T7RNAPNSV) (PU et al., “Multidimensional Control of Cas9 by Evolved RNA Polymerase- Based Biosensors,” ACS Chem. Biol. 13:431 437 (2018), which is hereby incorporated by reference in its entirety) translationally fused to a nanobody (NB), another encoding the C- terminal portion (residues 180-883) of T7RNAP (T7RNAPC) translationally fused to a separate NB, and the reporter plasmid encoding [3-galactosidase (LacZ) under the regulation of a T7 promoter (pT7LacZ). Anti-eGFP NB sequences were codon optimized for E. coli and cloned into plasmids to create a C-terminal fusion on the T7RNAPNSV fragment and an N-terminal fusion on the T7RNAPCfragment. LaG2 and LaG14 were chosen (Fridy et al., “A Robust Pipeline for Rapid Production of Versatile Nanobody Repertoires,” Nat Methods 11: 1253- 1260 (2014), which is hereby incorporated by reference in its entirety) as the first NBs to test since their epitopes had little overlap (FIG. 7), a feature that was predicted to be important to allow both NBs to bind simultaneously. The same 14 amino acid (AA) flexible linker sequence was used on both plasmids to fuse the polymerase to the nanobody. The conversion of the yellow substrate chlorophenol red P-D-galactopyranoside (CPRG) to the purple product chlorophenol red (CPR) was monitored via absorbance at 580 nm to assess sensor functionality. To execute the assay, the T7RNAP fragment fusions were first “pre-expressed” in a CFE reaction step for one hour. Then, reporter plasmid, CPRG, and protein-containing sample were added. Pre-expression of the T7RNAP fragments prior to the final sensing reaction decreasesthe detection time of the assay. To control for the impacts of the addition of purified target protein and storage buffer on CFE, control reactions with either pure buffer or a separate unrelated protein added in place of eGFP target were used.

[0177] FIG. 1C shows that the addition of eGFP to the TLISA reaction increases the rate of LacZ expression relative to both the buffer and unrelated protein controls, demonstrating TLISA functionality for sensing proteins. The pigment-based reporter mechanism enables semi-quantitative result interpretation by naked eye observation of reaction color (FIG. ID). Yellow and orange colors that differ by more than 0.2 A.U. are visually distinguishable, but this threshold is closer to 0.5 A.U. for red shades (FIG. 8). Higher concentrations of eGFP leads to faster production of LacZ and thus faster color change (FIG. ID and FIG. 9). This specific sensor was able to detect 100 - 500 nM eGFP. It is also shown that TLISA reactions are robust to variation across lysate batches and to reactions on different days (FIGS. 10A-10B).

[0178] Table 3 shows a list of example sequences of T7 / RNAP fragments used in accordance with this disclosure.Table 3. Sequences of the T7RNAP fragments used in this study.(a) - Pu et al., “Evolution of a Split RNA Polymerase as a Versatile Biosensor Platform,” Nat Chem Biol 13:432^438 (2017), which is hereby incorporated by reference in its entirety(b) - Pu et al., “Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors,” ACS Chem. Biol. 13 :431 437 (2018), which is hereby incorporated by reference in its entirety(c) - Pu et al., “Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors,” ChemBioChem 20:547-1553 (2019), which is hereby incorporated by reference in its entiretyExample 2 - Flexibility of the TLISA Platform

[0179] To demonstrate the modularity of TLISA to work with different nanobodies, new anti- eGFP NB sequences were fused to both the T7RNAPNSVand T7RNAPCfragments and tested for eGFP detection. To facilitate the comparison of the resulting sensors, the area between curves (ABC) (see Materials and Methods) were used as a quantitative metric of sensor quality, where ABC £ 0 indicates no protein detection and higher ABC indicates more visually distinct responses for a longer period of time — and thus a better sensor. TLISA is rather robust to changes in NB sequence, with several combinations of anti-eGFP NBs producing functional biosensors without any optimization of reaction conditions (i.e., plasmid concentrations and ratios) (FIG. 2A and FIG. 11). It is noted that the T7RNAPCfragments used here contained mutations that propagated during cloning, and these mutations actually improved sensor quality in some instances (Table 4 and FIGS. 12A-12D).

[0180] During this study, and after most of the data were collected, mutations in the gene for T7RNAPC were noticed on several plasmids. Table 4 indicates which mutations were present on each plasmid used to generate the data in every figure. None of the plasmids used in this study have mutations in the NB, MB, or DARPin sequences. Additionally, there were no mutations in any plasmids encoding for the T7RNAPN or T7RNAPNev fragments. Plasmids not listed in Table 4 did not have any mutations.

[0181] After identifying mutations in these plasmids, the mutations on three NB-T7RNAPC plasmids were corrected to test how these mutations impact TLISA functionality. Correcting the mutations on the LaG2-T7RNAPC plasmid resulted in a dysfunctional sensor (FIG. 12A). The reporter plasmid concentration was decreased to reduce leak but still did not observe any eGFP detection under any conditions (FIG. 12B). For the eGFP sensor that uses a MB fusion to the T7RNAPC (T7RNAPNev-LaG2 / GS2-T7RNAPC), correcting the plasmid mutations improved the rate of reaction, and was still able to detect 500 nM eGFP (FIG. 12C). The eGFP sensor that uses a DARPin fusion to the T7RNAPC (T7RNAPNev-LaG2 / 3G86.32-T7RNAPC) also failed to detect eGFP after the mutations were corrected (FIG. 12D). In summary, while specific sensors can be more or less robust to these mutations, reversion of mutation VI 18M was most often deleterious and mutation G537R was generally less disruptive to performance.Table 4. Summary of plasmids containing mutations used in this study.

[0182] These results reveal interesting characteristics of the TLISA platform. First, it was expected that to detect monomeric proteins (such as eGFP), the NBs fused to each polymerase fragment would need different epitopes to prevent steric occlusion of one NB by the other, which would otherwise prevent T7RNAP reassembly and reporter expression. The epitopes of the six NBs used here can be organized into three groups based on the general location of their epitopes where LaG14 is in group I; Nbl, LaG19, LaG26, and LaG27 are in group II; and LaG2 is in group III (Fridy et al., “A Robust Pipeline for Rapid Production of Versatile Nanobody Repertoires,” Nat Methods 11:1253-1260 (2014) and Kubala et al., “Structural and Thermodynamic Analysis of the GFP:GFP-Nanobody Complex,” Protein Science 19:2389- 2401 (2010), both of which are hereby incorporated by reference in their entirety). The epitopes of LaG19, LaG26, and LaG27 overlap significantly (FIG. 7), which could explain why there were no functional sensors created from combinations of these NBs. Surprisingly, a sensor with two LaG2 NBs still yielded significant detection signal. Beyond potential shortcomings in existing epitope maps for these NBs, the mechanism for this signal remains unclear. It was also observed that for a given pair of NBs, the ABC could be quite different depending on which NB was on the N versus C fragment. This could be due to differences in protein folding caused by tertiary structure interactions between domains or by improved folding for certain NBs for either N- or C-terminal fusions (as the two fragments had their NBs fused on opposite termini). Lastly, many but not all combinations of NBs result in functional TLISA sensors, and the sensing quality and rate of reaction even for those sensors can vary greatly. Direct eGFP ELISA data confirm that all six anti-eGFP NBs can fold correctly in the CFE environment (FIG. 13). These results provide some guidance on which NBs may yield functional TLISA sensors, though it is imperfect. For example, LaG26 had one of the weakest responses in the direct ELISA and also typically had low ABC values in TLISA assays, but NB1 had some of the highest ABC values despite relatively low activity in the direct ELISA. Thus, there are multiple variables that determine whether a NB will prove functional in TLISA, and more exploration is needed to identify characteristics allowing de novo prediction of function. Moreover, it is noted that while all combinations tested in FIG. 2A were executed under identical reaction conditions, the performance of individual biosensors can often be further improved simply by optimizing the concentration of plasmid expressing LacZ (FIGS. 14A- 14C).

[0183] Next, it was shown that TLISA is compatible with different types of protein affinity domains. Two different synthetic protein binding scaffolds were implemented as TLISA affinity domains: a monobody and a DARPin (designed ankyrin repeat protein). Monobodies (MBs) are small (ca. 10 kDa) binding proteins based on the fibronectin type III domain. DARPins are based on natural ankyrin repeat protein scaffolds. A new eGFP biosensor using the GS2 MB (Koide et al., “Teaching an Old Scaffold New Tricks: Monobodies Constructed Using Alternative Surfaces of the FN3 Scaffold,” J Mol Biol 415:393-405 (2012), which is hereby incorporated by reference in its entirety) (FIG. 2B) and another sensor using the 3G86.32 DARPin (Brauchle et al., “Protein Interference Applications in Cellular and Developmental Biology Using DARPins that Recognize GFP and mCherry,” Biology Open 3:1252-1261 (2014), which is hereby incorporated by reference in its entirety) (FIG. 2C) were engineered simply by switching them into the T7RNAPCfusion and maintaining all reaction conditions. These results show that TLISA can be robust to different sizes and scaffolds of affinity domains, and that different types of domains can be mixed and matched in one sensor for a plug-and-play platform suitable for rapid re-engineering.

[0184] A list of exemplary sequences of nanobodies, monobodies, and DARPins are listed in Table 5.Table 5. Sequences of nanobodies, monobodies, and DARPins used in this disclosure.(a ’)- Kubala et al., “Structural and Thermodynamic Analysis of the GFP:GFP-Nanobody Complex,” Protein Science 19:2389-2401 (2010), which is hereby incorporated by reference in its entirety(b ) - Fridy et al., “A Robust Pipeline for Rapid Production of Versatile Nanobody Repertoires,” Nat Methods 11 : 1253-1260 (2014), which is hereby incorporated by reference in its entirety(c’) - Koide et al., “Teaching an Old Scaffold New Tricks: Monobodies Constructed Using Alternative Surfaces of the FN3 Scaffold,” J Mol Biol 415:393-405 (2012), which is hereby incorporated by reference in its entirety(d) - Brauchle et al., “Protein Interference Applications in Cellular and Developmental Biology Using DARPins that Recognize GFP and mCherry,” Biology Open 3:1252-1261 (2014), which is hereby incorporated by reference in its entirety(e’) - Koenig et al., “Structure-Guided Multivalent Nanobodies Block SARS-CoV-2 Infection and Suppress Mutational Escape,” Science 371 :eabe6230 (2021), which is hereby incorporated by reference in its entirety(f) - Chonira et al., “A Potent and Broad Neutralization of SARS-CoV-2 Variants of Concern by DARPins,” Nat Chem Biol 19:284-291 (2023), which is hereby incorporated by reference in its entirety( ■ Ma et al., “Development of Nanobody-Based Flow Injection Chemiluminescence Immunoassay for Sensitive Detection of Human Prealbumin,” Biosensors and Bioelectronics 61 :165-171 (2014), which is hereby incorporated by reference in its entirety

[0185] Next, the influence of the length of the linker that connects the polymerase fragments to the affinity domains was investigated. The TLISA sensors in FIGS. 1A-1D and FIGS. 2A- 2C all used a previously reported (Pu et al., “Evolution of a Split RNA Polymerase as aVersatile Biosensor Platform,” Nat Chem Biol 13:432—438 (2017), which is hereby incorporated by reference in its entirety) 14 AA flexible linker to translationally fuse each affinity domain to the T7RNAP fragments. Choosing one representative eGFP sensor (T7RNAPNcv-NBl / LaG2-T7RNAPc), linker lengths were altered from the original 14 AAs to either 7 or 28 AAs (first 7 AAs of the linker or the entire 14 AA sequence repeated; Table 6) for each polymerase fragment and tested all possible combinations for eGFP detection. Linker length affected detection functionality differently for the two polymerase fragments (FIG. 3A). Using a 28 AA linker on the T7RNAPNCV fragment abrogated detection in all combinations, while longer linkers on the T7RNAPCfragment generally yielded strong detection. The best combination used a 7 AA linker on the T7RNAI\... fragment and a 28 AA linker on the T7RNAPCfragment (FIG. 3B). Again, it is suspected that these observations could be due to differences in protein folding and stability for the fused constructs. While these results may not generalize to all combinations of NBs and their respective epitope locations, it was found generally that using the same 14 AA linker is sufficiently modular to work for many applications, but further tuning of performance for the same pair of NBs is possible via linker optimization if desired.Table 6. Sequences of the flexible amino acid linkers used in this study.Linker 14 was taken from literature as a functional linker sequence for fusing affinity domains to the split T7 RNAP (Pu et al., “Evolution of a Split RNA Polymerase as a Versatile Biosensor Platform,” Nat Chem Biol 13:432-438 (2017), which is hereby incorporated by reference in its entirety). Linker7 is the first 7 AAs of Linkerl4. Linker28 is Linkerl4 doubled.

[0186] The impacts of changing the terminus for the NB fusion for each polymerase fragment was also investigated. All experiments thus far used the C-terminus of T7RNAPNCVand N- terminus of T7RNAPCfor fusions, based on previous reports for in vivo split T7RNAP biosensors (Pu et al., “Evolution of a Split RNA Polymerase as a Versatile Biosensor Platform,” Nat Chem Biol 13:432— 438 (2017) and Pu et al., “Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors,” ACS Chem. Biol. 13 :431 — 437 (2018), both ofwhich are hereby incorporated by reference in their entirety). However, as has already been shown, functionality is dependent on the local fusion context, so changing fusion locations could potentially yield even better sensors. All four combinations of fusion termini with the split T7RNAP system were tested using the wt version of both fragments; this wt version of the T7RNAPNfragment has not been evolved to minimize spontaneous reassembly, and thus provides a way to assess whether functional polymerase can reassemble at all in a CFE system. It was found that both N- and C- terminal fusions to wt T7RNAPNyield reassembly of functional polymerase, while T7RNAPCdoes not tolerate C-terminal fusions (FIG. 15), consistent with previous reports. Pu et al., “Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors,” ChemBioChem 20:547-1553 (2019), which is hereby incorporated by reference in its entirety. A second, mutated T7RNAPCfragment (T7RNAPCL2A) was then tested whose mutations enable C-terminal fusions (z<7.); spontaneous reassembly in a CFE system with aNB fused to the C-terminus of the T7RNAPC,L2A fragment (FIG. 15) was validated even though this was not possible with the original T7RNAPCfragment.

[0187] Assessment of TLISA functionality for NB fusions on different termini for T7RNAPNevand T7RNAPC,L2A showed some degree of robustness to these changes. Although the wt T7RNAPNtolerated both N- and C- terminal fusions, no detection and minimal baseline activity was observed when fusing NBs to the N- terminus of T7RNAPNev (FIG. 3C). This loss of activity may be due to protein folding differences between the wt T7RNAPNand T7RNAPNCV. On the other hand, functional eGFP biosensors with NB fusions to both the N- and C- terminus of T7RNAPC,L2A (FIG. 3D) were demonstrated. An N-terminal NB fusion on T7RNAPC.L2A resulted in a functional eGFP sensor using the established reaction conditions without any optimization while the pT7LacZ reporter plasmid concentration needed to be increased to observe eGFP detection when using the C-terminal NB fusion on the T7RNAPC,2LA. Thus, future TLISA designs could use selection of termini for fusions as another possible avenue for optimization.Example 3 - TLISA can easily be reengineered to detect different antigens

[0188] Next, it was shown that the TLISA platform can be used to detect a different protein antigen. The modular design nature of TLISA makes this simple: new plasmids were cloned with anti-mCherry NB fusions to the polymerase fragments to create a new TLISA sensor formCherry (FIG. 4A). The epitopes of these NBs were not characterized at the time of selection, so of the available NBs the four with the lowest Kdvalues were chosen (Fridy et al., “A Robust Pipeline for Rapid Production of Versatile Nanobody Repertoires,” Nat Methods 11: 1253- 1260 (2014), which is hereby incorporated by reference in its entirety) and validated proper CFE with an ELISA (FIG. 16). mCherry detection was tested for using all 16 possible nanobody combinations. The best-performing sensor, T7RNAPNcv-LaM4 / LaM2-T7RNAPc, had an ABC of 54.9 (FIG. 4A) and an LOD of 50 nM mCherry (FIGS. 17A-17B). Similar to eGFP sensors, many different combinations of anti-mCherry nanobodies can be used to produce functional mCherry sensors (FIG. 4B and FIG. 18). The epitopes of LaM2, LaM4, and LaM6 were later reported, revealing that the epitopes of LaM2 and LaM6 overlap significantly while the epitope of LaM4 is separate (Cong et al., “Schellenberg, High-Efficiency Recombinant Protein Purification Using mCherry and YFP Nanobody Affinity Matrices,” Protein Science 31 :e4383 (2022), which is hereby incorporated by reference in its entirety) and thus explaining why sensors combining LaM2 and LaM6 yielded no detection. It was also shown that TLISA can use reporter proteins other than LacZ, implementing superfolder GFP (sfGFP) as a reporter protein for an mCherry TLISA now that eGFP was no longer the target antigen (FIGS. 19A-19B). Implementing reporter proteins other than LacZ also enables the use of commercial CFE systems, such as the myTXTL Sigma 70 kit (Garamella et al., “The All E. Coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology,” ACS Synth. Biol. 5:344-355 (2016) and Garenne et al., “The all-E. coliTXTL toolbox 3.0: New Capabilities of a Cell-Free Synthetic Biology Platform,” Synthetic Biology 6:ysab017 (2021), both of which are hereby incorporated by reference in their entirety), to build TLISA sensors. TLISA is robust to variations between different CFE systems, and had no significant difference in dynamic range when sensing 200 nM mCherry in an in-house prepared CFE system and in the myTXTL Sigma 70 system (FIGS. 19B-19C).Example 4 - Rapid development of clinically relevant biosensors

[0189] Next, it was shown that it is straightforward to apply the TLISA platform for detection of clinically relevant targets. A biosensor was first engineered to detect the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein. NBs VHHE and VHHV were chosen due to their high binding affinities and nonoverlapping epitopes (FIG. 20) (Koenig et al., “Structure-Guided Multivalent NanobodiesBlock SARS-CoV-2 Infection and Suppress Mutational Escape,” Science 371 :eabe6230 (2021), which is hereby incorporated by reference in its entirety). NB sequences were codon optimized, cloned into T7RNAP fragment plasmids as translational fusions, and tested for the ability to detect soluble RBD. By fusing VHHE to T7RNAPNCVand VHHV to T7RNAPC, a TLISA RBD biosensor was successfully engineered and maximum signal-to-noise in 35 minutes was observed (FIG. 5A). Significant RBD detection was also observed when exchanging the fragments to which the NBs were fused (i.e., fusing VHHV to T7RNAPNSVand VHHE to T7RNAPc), though the signal was not as strong (FIG. 21). Since it was previously shown that DARPins can be efficiently incorporated into the TLISA platform (FIG. 2C), an existing DARPin (FSR22) was selected (Chonira et al., “A Potent and Broad Neutralization of SARS-CoV-2 Variants of Concern by DARPins,” Nat Chem Biol 19:284-291 (2023), which is hereby incorporated by reference in its entirety) against the RBD and fused it to T7RNAPNCVto generate another functional TLISA sensor (FIG. 5B), which yields clear visual detection of 500 nM RBD in 45 minutes (FIG. 22A). This TLISA sensor has lower leak than the dual-NB version, and reducing the pT7LacZ concentration lowers the leak even further, which could aid in visual interpretability (FIG. 22B). While focus was on the detection of soluble RBD for this proof-of-concept, verification was completed that this TLISA can also detect 150 nM of full- size SARS-CoV-2 S protein, with visually distinct results in 45 minutes (FIGS. 23A-23B). The current LOD of this biosensor is 200 nM RBD (FIGS. 24A-24B); while this is greater than the LOD of commercialized rapid antigen tests (Stanley et al., “Limit of Detection for Rapid Antigen Testing of the SARS-CoV-2 Omicron and Delta Variants of Concern Using Live- Virus Culture,” Journal of Clinical Microbiology 60 :e00140-22 (2022), which is hereby incorporated by reference in its entirety) and would thus require further engineering for clinical deployment, it has nonetheless been shown that TLISA enables quick and easy creation of biosensors against targets of clinical interest without extensive optimization. Additionally, FIG. 5A and FIG. 5B show that both RBD biosensors are robust to day-to-day experimental variation.

[0190] A biosensor was then created for another clinically relevant target, transthyretin (TTR). Serum TTR has historically been used as a biomarker to determine overall nutritional status (Bharadwaj et al., “Malnutrition: Laboratory Markers vs Nutritional Assessment,” Gastroenterology Report 4:272-280 (2016), which is hereby incorporated by reference in its entirety). Malnutrition is the direct cause of about 300,000 deaths per year (Muller andKrawinkel, “Malnutrition and Health in Developing Countries,” CMAJ 173:279-286 (2005) and Drammeh et al., “Determinants of Household Food Insecurity and Its Association with Child Malnutrition in Sub-Saharan Africa: A Review of the Literature,” Current Research in Nutrition and Food Science Journal 7:610-623 (2019), both of which are hereby incorporated by reference in their entirety), primarily in the developing world. Thus, a TLISA biosensor for TTR would provide an inexpensive, equipment-free, and potentially globally impactful POC assay for malnutrition. Such assays are sought after by government organizations such as the DHS Program, who execute nutritional surveys to determine which populations are in most need of nutritional intervention (Corsi et al., “Subramanian, Demographic and Health Surveys: A Profile,” International Journal of Epidemiology 4:1602-1613 (2012), which is hereby incorporated by reference in its entirety).

[0191] Because TTR is a homotetramer, it was posited that TLISA could work even with the same NB fused to both plasmids, simplifying sensor design. Thus, NB03 (Ma et al., “Development of Nanobody-Based Flow Injection Chemiluminescence Immunoassay for Sensitive Detection of Human Prealbumin,” Biosensors and Bioelectronics 61 :165-171 (2014), which is hereby incorporated by reference in its entirety) was chosen as the protein affinity domain for both T7RNAP fragments. The first TTR sensor that was constructed successfully detected TTR within 1 hour with robustness to day-to-day variation (FIG. 5C). The signal-to-noise ratio was relatively low, though, so some basic sensor optimization was attempted. The linker length was decreased from 14 AA to 7 AA on the T7RNAPNCV-NB03 fragment based on the eGFP sensor results (FIG. 3B). This resulted in a much faster reaction time, and the ABC increased from 6.7934 to 16.7577 (FIGS. 25A-25E). Decreasing the concentration of the pT7LacZ reporter plasmid from 0.12 nM to 0.05 nM to slow the reaction rate yielded a similar ABC but with less leak and thus with potentially improved visual interpretability (FIGS. 25A-25E). Healthy serum TTR ranges from 3 pM to 8 pM (Hood et al., “Update on Disease-Specific Biomarkers in Transthyretin Cardiac Amyloidosis,” Curr Heart Fail Rep 19:356-363 (2022), which is hereby incorporated by reference in its entirety), with lower levels indicating potential malnutrition. This TLISA biosensor has an LOD of 500 nM TTR, which is well below the healthy range of serum TTR, meaning that this assay can be useful when simply diluting samples such that the “effective” TTR concentration in the reaction falls within this range or by using molecular biology approaches to decrease the effective LOD of the assay.Example 5 - Towards point-of-care use

[0192] TLISA is also robust for detection in complex biological sample matrices that would be used in a clinical diagnostic device. The established reaction protocol remains the same, but the protein to be detected is spiked into a biological sample matrix that is added after the preexpression (FIG. 6A). For field deployment, pre-expression would be a part of the manufacturing process, not an end user step. Lysates enriched with NB-T7RNAP fragments can be used to eliminate the need for pre-expression, but pre-expression was used here to characterize many different sensors more efficiently. To account for increased RNAse activity in human serum and saliva, 1% Murine RNAse inhibitor was added per previously established protocols (Hunt et al., “Towards Detection of SARS-CoV-2 RNA in Human Saliva: A Paper- Based Cell-Free Toehold Switch Biosensor with a Visual Bioluminescent Output,” New Biotechnology 66:53-60 (2022), , which is hereby incorporated by reference in its entirety). For an mCherry sensor, either 10% or 20% pooled human saliva or pooled human serum spiked with target protein was added.

[0193] FIG. 6B shows successful detection of mCherry in all conditions. Plotted are final absorbance values at the time when the signal-to-noise ratio was the greatest (FIGS. 26A-26B). It was chosen to plot end-point absorbance values rather than the ABC values since single-time point absorbance values are what would be interpreted visually by users at the POC. The substantially lower final absorbance values observed when running reactions in serum versus defined aqueous mixtures are likely due to the binding of serum albumin to CPR eliciting a shift in the absorption spectrum. McNemey et al., “Point-of-Care Biomarker Quantification Enabled by Sample-Specific Calibration,” Science Advances 5:eaax4473 (2019), which is hereby incorporated by reference in its entirety. The addition of naproxen to reactions with CPRG in serum restores proper color (FIGS. 27A-27B).

[0194] Moreover, TLISA can detect clinically relevant human biomarkers in their corresponding complex sample matrices. To demonstrate this, function of a sensor targeting SARS-CoV-2 RBD (T7RNAPNCV-FSR22 / VHHV-T7RNAPC) after spiking in 20% human saliva was validated. The addition of 20% human saliva did not impact RBD detection (FIG. 6C and FIG. 26B). Towards the field-friendly vision, it was also confirmed that TLISA can detect 200 nM RBD (FIG. 28) and 150 nM of the full SARS-CoV-2 S protein (FIG. 6D) within a 20%human saliva matrix when reacted at room temperature (25 °C). The pigment-based reporter mechanism enables equipment-free result interpretation (FIG. 6E).

[0195] Finally, it was demonstrated that TLISA has the potential for use in minimally equipped settings by showing it retains function after lyophilization. Lyophilization of cell-free reactions helps enable stable room temperature storage of biosensors that can then be rehydrated and used at the POC. Lyophilized cell-free reactions using the PURExpress system retain transcription and translation activity after a year of room temperature storage. Pardee et al., “Paper-Based Synthetic Gene Networks,” Cell 159:940-954 (2014), which is hereby incorporated by reference in its entirety. While crude lysate-based systems are relatively less stable at room temperature after lyophilization, adding cryoprotectants can improve their room temperature shelf-life (Brookwell et al., “Development of Solid-State Storage for Cell-Free Expression Systems,” ACS' Synth. Biol. 12:2561-2577 (2023); Wilding et al., “Thermostable Lyoprotectant-Enhanced Cell-Free Protein Synthesis for On-Demand Endotoxin-Free Therapeutic Production,” New Biotechnology 53:73-80 (2019); and Warfel et al., “A Low- Cost, Thermostable, Cell-Free Protein Synthesis Platform for On-Demand Production of Conjugate Vaccines,” ACS Synth. Biol. 12:95-107 (2023), all of which are hereby incorporated by reference in their entirety). Lyophilized cell-free reactions can be stored and transported as pellets (Huang et al., “BioBits™ Explorer: A Modular Synthetic Biology Education Kit,” Science Advances 4:eaat5105 (2018), which is hereby incorporated by reference in its entirety) or embedded onto porous substrates like paper (Pardee et al., “Paper-Based Synthetic Gene Networks,” Cell 159:940-954 (2014) and Blum et al., “Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression,” ACS Synth. Biol. 10: 1116-1131 (2021), both of which are hereby incorporated by reference in their entirety). The TLISA reactions were lyophilized after the pre-expression step (FIG. 6A), just before sample would be added. First, it was confirmed that pre-expressed T7RNAP and split T7RNAP polymerase fragments can be lyophilized after pre-expression and properly rehydrated (FIGS. 29A and 29B). Then, it was shown that TLISA sensing reactions are robust to lyophilization; this was demonstrated with the T7RNAPNcv-LaM4 / LaM2-T7RNAPcmCherry sensor (FIG. 29C) as well as the SARS-CoV-2 RBD sensor (FIG. 29D). Finally, after adjusting RNAse inhibitor and pT7LacZ concentrations, detection of SARS-CoV-2 RBD in 20% human saliva from a lyophilized TLISA reaction (FIGS. 6F, 29E, 29F) was demonstrated. Taken together, rapiddetection of human biomarkers in biological matrices from lyophilized reactions demonstrates that TLISA is well-suited for future clinical diagnostic applications.Example 6 - Discussion of Examples 1-5

[0196] A novel, adaptable, and generalizable CFE biosensing platform for protein detection is developed and characterized. TLISA can detect different antigens using numerous combinations of nanobodies, monobodies, and DARPins, can be linked to different reporter proteins, and can implemented in both commercial and in-house prepared CFE formulations, demonstrating the truly modular nature of this platform. Importantly, TLISA is robust to both day-to-day experimental variation and variation between different batches of in-house prepared crude lysate with only minor changes in sensor performance. It also functions robustly at room temperature, in complex biological matrices, and after lyophilization, achieving room temperature colorimetric detection of SARS-CoV-2 RBD in human saliva in just one hour.

[0197] Compared to existing POC assays, TLISA presents improvements or competitive performance in many key features (Table 7).Table 7. Comparing conventional protein detection assays with TLISA.(a ”) - Klumpp-Thomas et al., “Standardization of ELISA Protocols for Serosurveys of the SARS-CoV-2 Pandemic Using Clinical and At-Home Blood Sampling,” Nat Commun 12: 113 (2021), which is hereby incorporated by reference in its entirety(b ”) - de Assis et al., “Cost-Effectiveness of Anti-SARS-CoV-2 Antibody Diagnostic Tests in Brazil. ,” PLoS One 17:e0264 l 59 (2022), which is hereby incorporated by reference in its entirety(c”) - Biby et al., “Rapid Testing for Coronavirus Disease 2019 (COVID- 19),” MRS Communications 12:12-23 (2022), which is hereby incorporated by reference in its entirety(d”) - Sun et al., “Protocols for Implementing an Escherichia Coli Based TX-TL Cell-Free Expression System for Synthetic Biology,” J Vis Exp e50762 (2013), which is hereby incorporated by reference in its entiretyLODs can depend on many factors and can vary greatly for a single type of assay. Costs provided do not include any labor costs. The cost for TLISA was determined using previously calculated reagent costs for lysate-based cell-free reactions and accounts for one test reaction and one control reaction. Ease of development takes into consideration the time required to create antigen-binding domains and implement into the assay. ELISAs and LFAs both use monoclonal antibodies, which have a long development timeline and thus add to the complexity of developing ELISAs and LFAs for new targets. TLISA uses non-antibody affinity domains, which are simpler to generate and engineer. User-friendliness considers the need for laboratory equipment and long incubation steps to run the assay.

[0198] In particular, while the LOD of the unoptimized TLISA assays here is not as low as the highly engineered and optimized LFAs and ELISAs, it presents significant advantages in terms of cost and modularity for rapid development and deployment of new sensors that is currently extremely complicated for techniques such as LFAs, while also being competitive for ease of use and time to results. While TLISA in a field-deployable form is semi-quantitative as compared to the full quantitation of an in-lab ELISA assay, it is more quantitative than an LFA. Moreover, if implemented as a lab assay in a plate reader with a fluorescent output, TLISA would be positioned for fully quantitative output, though in its current unoptimized form it would have a narrower dynamic range than an ELISA. It is thus positioned, even in this early stage, as a competitive technology for modular, at-scale, field-deployable use.

[0199] While TLISA shows promise to be an impactful POC diagnostic tool, performance improvements will be critical for future in-field deployment. Although many of the sensors presented here exhibited low leak and high signal in the presence of target antigen, some had less desirable signal-to-noise ratios. It is shown here that using different NB fusions, linker lengths, and reporter plasmid concentrations can help tune performance to achieve a more desirable output for a given application if further optimization is needed beyond initial characterization. High-throughput studies to identify trends between NB sequence and signal- to-noise ratio could provide particularly useful insights for de novo engineering of sensors for new targets. New strategies to decrease leak due to spontaneous RNAP reassembly (FIGS. 30A-30C) would also significantly improve sensitivity and usability in some applications. Forexample, testing different T7RNAPNev variants that have less spontaneous reassembly (Pu et al., “Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors,” ACS Chem. Biol. 13:431-437 (2018), which is hereby incorporated by reference in its entirety) than the variant used in this work could be an effective strategy, as could the testing of multiple candidate affinity domains in different orientations (as described in this work) and the use of protein folding and docking software to predict which fusions are most or least likely to spontaneously reassemble. Nonetheless, there are applications where sensitivity at levels similar to the TLISA examples presented here (e.g., the mCherry TLISA with an LOD of 50 nM (FIGS. 17A-17B) would be effective, such as for the detection of the inflammation biomarker C-reactive protein, whose elevated levels are defined as above 5 mg / L (corresponding to 40 nM) (Namaste et al., “Methodologic Approach for the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (B RIND A) Project,” Am J Clin Nutr 106:333S-347S (2017), which is hereby incorporated by reference in its entirety). Additionally, as previously mentioned, the current linear range of detection is narrower than that of an ELISA. Broadening this range would increase the potential application space of TLISA. Nonetheless, the dose responses shown here provide an important feature that standard lateral flow assays for POC protein detection lack.

[0200] Even beyond POC applications, though, TLISA could provide unique capabilities in clinical laboratories as well. Compared to ELISA assays that are widely used for measuring proteins, the TLISA workflow is much simpler and much less subject to inter-operator variability, which would improve reliability of results. In addition, at 10 pL the volume necessary for a TLISA assay is extremely small, which could provide significant advantages for a clinical assay as more measurements could be taken from the same sample. This benefit would be particularly impactful for neonatal care where daily blood draw volumes can be limited to just a few mL.

[0201] The initial characterization presented here has only begun to explore the potential breadth of this platform. The successful use of nanobodies, DARPins, and monobodies in TLISA suggests a substantial repertoire of potential affinity domain fusions. It is anticipated that many other scaffolds such as single-chain variable fragments (scFvs), affibodies, and anticalins might also be used to develop novel protein sensors. Furthermore, TLISA can potentially be used to engineer protein-mediated OR gates by using bispecific affinity domains, as well as AND gates by splitting the T7RNAP into additional fragments at previouslyidentified cut sites (Segall-Shapiro et al., “A ‘Resource Allocator’ for Transcription Based on a Highly Fragmented T7 RNA Polymerase,” Molecular Systems Biology 10:742 (2014), which is hereby incorporated by reference in its entirety). Finally, while the efforts were focused on sensing proteins so as to fill a significant gap in the field, using affinity domains against nonprotein antigens (e.g., small molecules, glycans, etc.) could expand TLISA to sense a variety of diverse analytes and give it an even broader potential impact.Example 7 - TLISA Glycan Detection

[0202] While efforts to date have focused on making TLISA a versatile protein sensing platform, in principle, TLISA could be used to detect diverse classes of analytes. Preliminary work suggests that TLISA could be used to detect cell surface glycans. Glycans (carbohydrates) are information-rich biomarkers used in clinical diagnoses of diabetes, cancers, and other diseases. Hu et al., “Chapter One - Glycan-based Biomarkers for Diagnosis of Cancers and Other Diseases: Past, Present, and Future” in Progress in Molecular Biology and Translational Science, L. Zhang, Ed. (Academic Press, 2019; vol. 162 of Glycans and Glycosaminoglycans as Clinical Biomarkers and Therapeutics - Part A, pp. 1-24, which is hereby incorporated by reference in its entirety. Glycans, which form complex layers on the cell surface, play a critical role in intercellular interactions and immune response. In cancer cells specifically, Globo-H is a prevalent and well-studied tumor-associated carbohydrate antigen (TACA) overexpressed in my cancerous cell lines such as MCF7 (breast), A549 (lung) and HCT116 (colon). Jing et al., “Super-Resolution Imaging of Cancer- Associated Carbohydrates Using Aptamer Probes. Nanoscale 11 :14879-14886 (2019) and Sigal et al., “The Role of Globo H and S SEA-4 in the Development and Progression of Cancer, and Their Potential as Therapeutic Targets,” Future Oncol 18:117-134 (2022), both of which are hereby incorporated by reference in their entirety. Clinical methods for glycan identification include purification via chromatography and detection using methods like mass-spectrometry or ELISA.

[0203] It is believed that TLISA can be used to detect native Globo-H quickly and easily on cell surfaces. The assay would run almost identically to the previously described TLISA protocol with some minor changes (FIG. 32A). In theory, only one unique nanobody or affinity domain would be necessary to develop the Globo-H TLISA because there would be many Globo-H antigens present on the cell surface such that there would not be prohibitivecompetition for the same epitope by the affinity domains on each T7RNAP fragment. The anti- Globo-H nanobody would be translationally fused to both the T7RNAPNCV and T7RNAPc fragments. The TLISA cell-free reaction would take place directly on top of the cells. This is advantageous over a typical ELISA assay because it only requires one wash step (to initially remove the media from the cells). Preliminary data using Globo-H positive A549 cells suggests that this is feasible (FIG. 32B). More experimentation is required to assure reproducibility of the results.

[0204] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the application and these are therefore considered to be within the scope of the application as defined in the claims which follow.

Claims

WHAT IS CLAIMED:

1. A method for detecting an analyte level in a sample, the method comprising: providing a first RNA polymerase fragment comprising a first analyte affinity domain bound thereto at an end of the first RNA polymerase fragment, the first analyte affinity domain configured to bind to a target analyte; providing a second RNA polymerase fragment comprising a second analyte affinity domain bound thereto at an end of the second RNA polymerase fragment, the second analyte affinity domain configured to bind to a target analyte; providing a biological sample comprising a target analyte; adding the biological sample comprising the target analyte to the first RNA polymerase fragment and second RNA polymerase fragment to form a reaction mixture; and subjecting the reaction mixture to a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output.

2. The method of claim 1, wherein the analyte is a protein, a glycan, a small molecule, a virus particle, or a combination thereof.

3. The method of claim 1 or claim 2, wherein the first analyte affinity domain is a protein affinity domain, or the second analyte affinity domain is a protein affinity domain, or both the first and second analyte affinity domains are protein affinity domains.

4. The method of claim 1, wherein the first RNA polymerase fragment and the second RNA polymerase fragment are configured to have minimal spontaneous reassembly.

5. The method of any of the preceding claims, wherein, when the first RNA polymerase fragment and the second RNA polymerase fragment are forcibly colocalized upon binding of the first and second affinity domains to the target analyte, thus driving a reassembly of the first RNA polymerase fragment and the second RNA polymerase fragment, turning on a reporter gene, and generating the measurable output.

6. The method of any of the preceding claims, wherein the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, mucus, lymph, interstitial fluid, cerebrospinal fluid, pus, breast milk, and amniotic fluid.

7. The method of any of the preceding claims, wherein the first fragment, second fragment, and biological sample are added into a cell-free expression mixture, the mixture comprising lysates of cells capable of executing transcription and translation.

8. The method of any of the preceding claims, wherein the first analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

9. The method of any of the preceding claims, wherein the first analyte affinity domain comprises a nanobody.

10. The method of any of the preceding claims, wherein the first analyte affinity domain comprises a monobody.

11. The method of any of the preceding claims, wherein the first analyte affinity domain comprises a DARPin.

12. The method of any of the preceding claims, wherein the second analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

13. The method of any of the preceding claims, wherein the second analyte affinity domain comprises a nanobody.

14. The method of any of the preceding claims, wherein the second analyte affinity domain comprises a monobody.

15. The method of any of the preceding claims, wherein the second analyte affinity domain comprises a DARPin.

16. The method of any of the preceding claims, wherein the first analyte affinity domain is selected from the group consisting ofNB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

17. The method of any of the preceding claims, wherein the second analyte affinity domain is selected from the group consisting of NB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

18. The method of any of the preceding claims, wherein the first analyte affinity domain and the second analyte affinity domain comprise minimally overlapping epitopes configured to allow the first analyte affinity domain and the second analyte affinity domain to bind simultaneously to the target analyte.

19. The method of any of the preceding claims, wherein the first RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

20. The method of claim 19, wherein the first RNA polymerase fragment comprises a split T7 RNA polymerase.

21. The method of claim 20, wherein the first RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPC.L2A.

22. The method of any of the preceding claims, wherein the second RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

23. The method of claim 22, wherein the second RNA polymerase fragment comprises a split T7 RNA polymerase.

24. The method of claim 23, wherein the second RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPCX2A.

25. The method of any of the preceding claims, wherein the measurable output comprises absorbance, fluorescence, luminescence, or a combination thereof.

26. The method of any of the preceding claims, wherein the measurable output comprises absorbance, fluorescence, or luminescence at 580 nm.

27. The method of any of the preceding claims further comprising: providing a linker sequence in the first RNA polymerase fragment, the second RNA polymerase fragment, or in both the first and second RNA polymerase fragments.

28. The method of claim 27, wherein the linker sequence is between 5 and 30 amino acids in length.

29. The method of claim 27 or 28, wherein the linker sequence comprises the amino acid sequences SEQ ID NOS: 104-106.

30. The method of any of the preceding claims, wherein the first RNA polymerase fragment is lyophilized, the second RNA polymerase fragment is lyophilized, or both the first RNA polymerase fragment and the second RNA polymerase fragment are lyophilized.

31. A kit for detecting an analyte level in a sample, said kit comprising: a first RNA polymerase fragment comprising a first analyte affinity domain bound thereto at an end of the first RNA polymerase fragment, the first analyte affinity domain configured to bind to a target analyte; and a second RNA polymerase fragment comprising a second analyte affinity domain bound thereto at an end of the second RNA polymerase fragment, the second analyte affinity domain configured to bind to a target analyte.

32. The kit of claim 31 , wherein the analyte is a protein, a glycan, a small molecule, a virus particle, or a combination thereof.

33. The kit of claim 31 or claim 32, wherein the first analyte affinity domain is a protein affinity domain, or the second analyte affinity domain is a protein affinity domain, or both the first and second analyte affinity domains are protein affinity domains.

34. The kit of any of claims 31-33, wherein the first RNA polymerase fragment and the second RNA polymerase fragment are mixed with a biological sample to form a reaction mixture.

35. The kit of claim 34, wherein the reaction mixture is subject to a condition under which the first RNA polymerase fragment and second RNA polymerase fragment reassemble and produce a measurable output.

36. The kit of claim 35, wherein the first RNA polymerase fragment and the second RNA polymerase fragment are configured to have minimal spontaneous reassembly when subject to said condition.

37. The kit of claim 35, wherein, when the first RNA polymerase fragment and the second RNA polymerase fragment are forcibly colocalized upon binding of the first and second affinity domains to the target analyte, thus driving a reassembly of the first RNA polymerase fragment and the second RNA polymerase fragment, turning on a reporter gene, and generating the measurable output.

38. The kit of any of claims 31-37, wherein the first fragment, second fragment, and biological sample are added into a cell-free expression mixture, the mixture comprising lysates of cells capable of executing transcription and translation.

39. The kit of any of claims 31-38, wherein the first analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment,a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

40. The kit of any of claims 31-39, wherein the first analyte affinity domain comprises a nanobody.

41. The kit of any of claims 31-40, wherein the first analyte affinity domain comprises a monobody.

42. The kit of any of claims 31-41, wherein the first analyte affinity domain comprises a DARPin.

43. The kit of any of claims 31-42, wherein the second analyte affinity domain comprises a nanobody, a monobody, a designed ankyrin repeat proteins (DARPin), an antibody fragment, a single-chain variable fragment (scFv), a fragment antigen-binding region (Fab), or a combination thereof.

44. The kit of any of claims 31-43, wherein the second analyte affinity domain comprises a nanobody.

45. The kit of any of claims 31-44, wherein the second analyte affinity domain comprises a monobody.

46. The kit of any of claims 31-45, wherein the second analyte affinity domain comprises a DARPin.

47. The kit of any of claims 31 -46, wherein the first analyte affinity domain is selected from the group consisting of NB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

48. The kit of any of claims 31-47, wherein the second analyte affinity domain is selected from the group consisting ofNB03, NB1, LaG2, LaG14, LaG19, LaG26, LaG27, GS2, LaM2, LaM3, LaM4, LaM6, VHHE, VHHV, FSR22, and 3G86.32.

49. The kit of any of claims 31-48, wherein the first analyte affinity domain and the second analyte affinity domain comprise minimally overlapping epitopes configured to allow the first analyte affinity domain and the second analyte affinity domain to bind simultaneously to the target analyte.

50. The kit of any of claims 31-49, wherein the first RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

51. The kit of any of claims 31-50, wherein the first RNA polymerase fragment comprises a split T7 RNA polymerase.

52. The kit of any of claims 31-51, wherein the first RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPC.L2A.

53. The kit of any of claims 31-52, wherein the second RNA polymerase fragment is derived from a single-subunit RNA polymerase or a multi-subunit RNA polymerase.

54. The kit of any of claims 31-53, wherein the second RNA polymerase fragment comprises a split T7 RNA polymerase.

55. The kit of any of claims 31-54, wherein the second RNA polymerase fragment comprises T7RNAPN, T7RNAPnev , T7RNAPC, or T7RNAPC.L2A.

56. The kit of claim 35, wherein the measurable output comprises absorbance, fluorescence, luminescence, or a combination thereof.

57. The kit of claim 56, wherein the measurable output comprises absorbance, fluorescence, or luminescence at 580 nm.

58. The kit of any of claims 31-57 further comprising: providing a linker sequence in the first RNA polymerase fragment, the second RNA polymerase fragment, or in both the first and second RNA polymerase fragments.

59. The kit of claim 58, wherein the linker sequence is between 5 and 30 amino acids in length.

60. The kit of claim 58 or claim 59, wherein the linker sequence comprises the amino acid sequences SEQ ID NOS: 104-106.

61. The kit of any of claims 31-60, wherein the first RNA polymerase fragment is lyophilized, the second RNA polymerase fragment is lyophilized, or both the first RNA polymerase fragment and the second RNA polymerase fragment are lyophilized.

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