Microfluidic systems and methods for assessing protease activity

Abstract

Protease activity can be assessed using a microfluidic system that comprises a microfluidic device and solid support, wherein the solid support comprises a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises a protease domain, a protease activation sequence, and a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to the first surface of the solid support. Protease activity can be assessed in a microfluidic system by reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises a protease domain and a protease activation sequence; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; reacting the activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; and detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

Description

Attorney Docket No.048536-800001WO / SF2025-027-2-PCT MICROFLUIDIC SYSTEMS AND METHODS FOR ASSESSING PROTEASE ACTIVITY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to US Application No.63 / 716,978 filed November 6, 2024, and US Application No.63 / 686,435 filed August 23, 2024, the disclosures of which are incorporated by reference herein in their entirety. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under OD033413 and U19 AI171110, awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE

[0003] A Sequence Listing in an XML file having the title “048536-800001WO- SequenceListingST26,” having 8,766 bytes and created on August 18, 2025, is incorporated by reference herein in its entirety. BACKGROUND

[0004] A major challenge in antiviral development is inevitable resistance, especially with RNA viruses like SARS-CoV-2. Understanding how mutations correlate to changes in protein function relative to inhibition is crucial in the ability to predict potential resistance mechanisms. While proteases have been model systems for understanding enzyme catalysis for decades through focused in vitro studies, it remains challenging to develop a mechanistic understanding of protease function beyond the active site. For example, substitutions in the local S1 pocket are not sufficient to interconvert the specificities of mammalian trypsin and chymotrypsin, which requires additional changes in distal loops. Furthermore, resistance in HIV protease to inhibitors can result from mutations distal to the active site, making it challenging to predict potential resistance mutations. Thus, to understand diverse protease functions, our models need to include the effects of residues beyond the active site.^

[0005] Despite its quantitative power, measurement of protease function through traditional biochemistry is labor intensive, typically involving cellular expression, purification, and activity measurement in a low-throughput manner. High-throughput display methods for proteasescreening (e.g., Denard, et al., Acs Synth Biol 10, 63–71 (2021) and Packer et al, Nat. Commun. 8, 956 (2017)) have identified select protease variants with increased turnover rates and drug resistance. Deep mutational scanning methods in yeast have measured the effects of all single mutants of the SARS-CoV-2 main protease (Mpro) on growth and intracellular cleavage assays. See Flynn, et al., Elife 11, e77433 (2022). Despite their throughput, these cell-based methods alone cannot biochemically dissect protease variants, requiring low-throughput methods for subsequent characterization (typically less than 10, but as high as 100 variants), and are limited in their ability to systematically control conditions (e.g., efficient export of drug-like molecules in yeast limits inhibition assays in this system).

[0006] Two of the^most significant drug targets for SARS-CoV-2 are Mproand^ Plproand have been primary targets of several small molecule therapeutic campaigns. Targeting viral proteases will directly block the processing of the viral polyprotein into the functional viral proteins. Paxlovid, a combination of two small molecule therapeutics (nirmatrelvir and ritonavir) has shown high efficacy against SARS-CoV-2 emerging variants and subvariants, in particular the sublineages of Omicron in which Mprohas point mutation P132H. Through genomic data, resistance mutations in SARS-CoV-2 Mprohave been found in strains circulating the population.

[0007] There is a need in the art for high throughput assays to detect, quantify, and characterize protease variants. Provided herein are, inter alia, solutions to these and other needs in the art. BRIEF SUMMARY

[0008] Provided herein is a solid support comprising a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises a protease domain, a protease activation sequence, and a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to the first surface of the solid support.

[0009] Provided herein is a solid support comprising a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises (i) a protease domain, (ii) a detectable N-terminal tag, (iii) a protease activation sequence, wherein the protease activation sequence links the detectable N-terminal tag to the protease domain, and (iv) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support.

[0010] Provided herein is a microfluidic system comprising a microfluidic device and a solidsupport as described herein.

[0011] Provided herein is a method for assessing protease activity in a microfluidic system comprising: (a) reacting an activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; wherein the activated protease fusion protein comprises a protease domain bound to a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support; and (b) detecting the detectable signal, thereby detecting protease activity in the microfluidic system

[0012] Provided herein is a method for assessing protease activity in a microfluidic system comprising: (a) reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a protease activation sequence, wherein the protease activation sequence is bound to the protease domain, and (iii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; (b) contacting the activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; and (d) detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

[0013] Provided herein is a method for assessing protease activity in a microfluidic system comprising: (a) reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a detectable N-terminal tag, (iii) a protease activation sequence, wherein the protease activation sequence links the detectable N- terminal tag to the protease domain, and (iii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; (b) contacting the activated protease fusion protein with a fluorogenic substrate, thereby generating a fluorescent signal; and (d) detecting the fluorescent signal, thereby detecting protease activity in the microfluidic system.

[0014] Provided herein is a glass slide (e.g., solid support). In embodiments, the glass slide is coated with a polydimethylsiloxane (PDMS) polymer on a first surface and a plurality of individual spatial areas in a microarray format are arranged on the PDMS polymer on the first surface of the glass slide (e.g., solid support); an avidin protein is bound to the PDMS polymer and to a biotinylated anti-tag protein antibody (e.g., biotin conjugated to anti-GFP antibody,anti-eGFP antibody, anti-mCherry antibody, anti-mNeonGreen antibody, anti-polyHis antibody, or anti-FLAG antibody); wherein each of the plurality of individual spatial areas comprises the avidin protein and a plurality of plasmids encoding an enzyme fusion protein; and wherein the enzyme fusion protein comprises an enzyme domain and a protein tag domain (e.g., fluorescent protein tag), wherein the protein tag domain is capable of binding to the biotinylated anti-tag protein antibody. The glass slide has a length between 45 mm and 55 mm, a width between 60 mm and 70 mm, and a depth of less than 0.17 mm; a plurality of individual spatial areas in a microarray format on a first surface of the glass slide; and an avidin protein bound to the first surface of the glass slide and bound to a biotinylated anti-tag protein antibody; wherein each of the plurality of individual spatial areas comprises the avidin protein and a plurality of plasmids encoding an enzyme fusion protein; wherein the enzyme fusion protein comprises an enzyme domain and a protein tag domain, wherein the protein tag domain is capable of binding to the biotinylated anti-tag protein antibody.

[0015] Provided herein is a method of attaching a biotinylated anti-tag protein antibody (e.g., biotin conjugated to anti-GFP antibody, anti-eGFP antibody, anti-mCherry antibody, anti- mNeonGreen antibody, anti-polyHis antibody, or anti-FLAG antibody) to a glass slide (e.g., solid support). The method includes contacting a biotinylated anti-tag protein antibody with an immobilized avidin protein wherein the immobilized avidin protein is attached to a PDMS layer on the glass slide.

[0016] Provided herein is a method of detecting enzyme activity in a microfluidic. The method includes contacting a library of enzyme fusion proteins forming part of a microfluidic system with an enzyme binder (e.g., a biomolecule such as a protein, a nucleic acid, or a small molecule that is capable of binding an enzyme) comprising a fluorescently-labeled moiety; wherein each enzyme fusion protein comprises an enzyme domain and a protein tag domain (e.g., fluorescent protein tag); wherein the protein tag domain is bound to a biotinylated anti-tag protein antibody and the biotinylated anti-tag protein antibody is bound to a first surface of a glass slide (e.g., solid support) through an avidin protein; detecting the fluorescently-labeled moiety, thereby detecting enzyme activity in the microfluidic system.

[0017] These and other embodiments of the disclose are described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG.1 illustrates a microfluidic system, consistent with implementaions of the current subject matter provided herein. A two-layer microfluidic device bound to a solid support (e.g.,glass slide) contains a plurality of chambers in which reactions occur. Fluidic inlets are disposed at one end of the device which are connected to external reagents, buffers, and the like. One or more outlets is positioned at the end of the fluidic device opposite the inlets. Outlets remove waste from the device. The process of forming the microfluidic system with a sample of interest is detailed in FIGS.2-4.

[0019] FIG.2 depicts an exemplary embodiment of preparing a solid support (e.g., glass slide) with plasmids arranged in a microarray configuration with known locations for use in a microfluidic system. In step 1, plasmid libraries are designed, parsed, and sequence verfied. In step 2, a polydimethylsiloxane (PDMS) polymer is prepared and deposited onto a solid support, such as glass, such that one surface of the solid support (e.g., glass slide) is coated with a layer of the PDMS polymer (i.e., a “PDMS layer”). In step 3, designed plasmid libraries are arrayed on the PDMS layer of the solid support (e.g., glass slide) at known locations using known printing techniques. The plasmid libraries are deposited onto the solid support (e.g., glass slide) in volumes of approximatey 400 picoliters and spaced to create individual spatial areas.

[0020] FIG.3 depicts an exemplary embodiment of a microfluidic device applied to a solid support (e.g., glass slide) to create a microfluidic system for high-throughput protease activity analysis. The microfluidic device is made of a plurality of chambers disposed within the surface that contacts the PDMS layer on the solid support (e.g., a PDMS-coated glass slide). The microfluidic device is aligned with the solid support (e.g., glass slide) such that each of the individual spatial areas of plasmid library solution is trapped within a chamber of the microfluidic device. The magnified portion of the solid support (e.g., glass slide) illustrates the individual spatial areas of plasmid library deposited in a series of columns.

[0021] FIG.4 depicts the microfluidic device and solid support (e.g., glass slide) forming a microfluidic system. Once the microfluidic device and solid support (e.g., glass slide) are aligned, the components are baked so that the microfluidic device seals to the PDMS-coated solid support (e.g., glass slide). The magnified area illustrates a plurality of sample chambers in fluid communication with a plurality of reaction chambers. The sample chambers are aligned over the plasmid libraries

[0022] FIG.5 is a picture of the microfluidic system containing the chambers and fluid connections within. The microfluidic system contains a total number of 1792 chambers between the sample chambers and the reaction chambers. A network of flow channels connects the sample chambers with the reaction chambers, and fluid movement between the channels is controlled by valves connected to control channels. As pressure within the control channelschanges, the valves open and close permitting fluid to move through the device. Button (B), sandwich (S), and neck (N) valves control the movement of fluid from the reaction chamber to the sample chamber. The magnified area of the microfluidic system illustrates the chambers, channels, valves, and other control features for the device.

[0023] FIG.6 is a schematic illustrating the steps to create an improved microfluidic system, as previously described in FIGS.2-5. At step I, a plasmid library microarray is created on a solid support (e.g., glass slide) where the prepared library is deposited onto the solid support (e.g., glass slide) in individual spatial areas. At step II, microfluidic devices are aligned over the plasmid library such that the chambers of the microfluidic device each capture one of the individual spatial areas. At step III, the components are heated so that the microfluidic device seals onto the PDMS-coated glass slide via crosslinking between the PDMS layer on the solid support (e.g., glass slide) and the PDMS of the device and the individual spatial areas are sealed within chambers of the microfluidic device. The microfluidic device contains a plurality of solution inlets and valve control inlets at one end. The solution inlets provide buffers, reagents, etc., into the device and chambers, and the valve control inletes connect to the control channels. As pressure is controlled at the valve control inlets, the valves open and close accordingly. The magnified area of the chambers illustrates the plasmid library within the sample chamber. In the opposite reaction chamber, a fluorescent protein tag (e.g., antibody) is attached to the PDMS layer of the solid support (e.g., glass slide) through an avidin protein.

[0024] FIG.7: Development of a microfluidic-based protease kinetic assay for the rapidcharacterization of activity and inhibition of Mpro variant libraries. Up to 1792 unique plasmidscan be printed and arrayed per microfluidic device with each chamber harboring an individual clone. Each Mpro-eGFP variant is expressed simultaneously using an in vitro transcription translation system and is immobilized using an anti-eGFP VHH. Captured enzyme is then activated using TEV to remove the N-terminal tag and generating the native N-terminus. For quality control, fluorescently-labelled anti-FLAG can be used to validate complete activation of Mpro. The activity of each Mprovariant can be quantified using an internally-quenched peptide substrate designed based on the NSP4-NSP5 cleavage site. HT-MEKproallows for the quantification of Michaelis-Menten parameters of each protease in terms of kcat, KM, and kcat / KM. Inhibition can also be quantified for each variant. HT-MEKprofacilitates the generation of extensive functional datasets for Mprovariants, offering insights into the role of individual mutations in both function and resistance. HT-MEKproefficiently characterizes a larger sequence space at an accelerated rate, overcoming time and resource constraints that often impede thescalability of traditional protein expression and characterization methods.

[0025] FIGS.8A-8G: Rational design of a functional Mpro-eGFP fusion compatible with microfluidic-based assays. FIG.8A: Mpro-eGFP fusion constructs contain five distinct domains: N-terminal tag, activation sequence, Protease Domain (SARS-CoV-2 Mpro), Gly-Ser Linker ((G4S)5), and Fluorescent Protein tag (eGFP). Primary engineering efforts focused on the N- terminal tag (GST, Myc, Triple-FLAG, and FLAG) and the activation sequence (auto-activation, enterokinase, and TEV). FIG.8B: Activation of WT Mproand active site control (C145A) from in gel fluorescence. Activation sequences for auto-activation (SEQ ID NO:4), TEV activation (SEQ ID NO:5), and TEV control (SEQ ID NO:6). FIG.8C: Reaction scheme of Rhodamine- 110-labelled fluorogenic substrate. Proteolytic activity of WT Mprounder different conditions quantified through fluorescence by plate reader. Inset contains representative progressive curves for activated WT Mproand no enzyme controls. FIG.8D: Expression levels for a range of N- terminal tags (FLAG, 3XFLAG, Myc, and GST) in comparison to a no template control. FIG. 8E: Activation of Mpro-eGFP controls on device visualized by anti-FLAG antibody conjugated to DyLight550. FIG.8F: Sample images of (left) immobilized enzyme, (center) immobilized anti-FLAG, and (right) fluorogenic product over time from Rh110 substrate for WT and C145A Mproand activation sequence controls (SEQ ID NO:7). FIG.8G: Example Rh110-labelled fluorogenic progress curve for Mprochambers containing WT enzyme and active site control (C145A) with and without nirmatrelvir (1 µM).

[0026] FIG.9: Mutational Effects on SARS-CoV-2 MproSubstrate Specificity. Heatmap showing log₂ fold change in initial reaction rates for SARS-CoV-2 Mprovariants relative to wild- type. Each column represents a distinct Mprovariant, and each row corresponds to one of nine SARS-CoV-2 polyprotein NSP cleavage sites (15 µM substrate, sub-saturating; two sites excluded due to poor activity across most variants). Shading indicates the magnitude and direction of rate changes, enabling identification of mutations that alter protease substrate specificity. For clarity, the heatmap for FIG.9 is presented over 3 pages, with the first page of FIG.9 showing the heading for each column that remains the same heading for the columns on the second and third pages of FIG.9. Similarly, the shading for the log2 fold at the bottom of the third page of FIG.9 applies to the first and second pages of FIG.9. DETAILED DESCRIPTION

[0027] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0028] “Microfluidic system” refers to the microfluidic device and the solid support (e.g., glass slide) working in combination to provide high-throughput reactions. Depending on the needs of the system, both the microfluidic device and the solid support (e.g., glass slide) can take on various configurations. As such, the microfluidic system is prepared according to the desired output.

[0029] “Microfluidic device” has its normal meaning in the art and refers to a device comprising a substrate with structures such as control channels, chambers, reaction chambers, valves, inlet and outlet ports, and the like. In embodiments a microfluidic device comprises two layers. In embodiments, a microfluidic device comprises a first layer which comprises a plurality of control channels, and a second layer which comprises a plurality of sample chambers, and a plurality of valves. The microfluidic devices described herein can take on numerous configurations. Microfluidic devices is well known in the art and described, for example, in Maerkl et al, (2007). Science 315 (5809): 233-237; Fordyce et al, (2010). Nature Biotechnology 28: 970-975; Aditham et al, (2021). Cell Systems 12 (2): 112-127); Markin et al, (2021). Science 373 (6553): eabf8761; Hastings et al, (2023). bioRxiv : 566946; US 2022 / 0235406; WO 2024 / 130109; US 11,187,702; US 2022 / 0050108; US 2022 / 0042995; US 7,501,245; US 2010 / 0311611; US 2002 / 0151078; EP 3677336; CA 2582476; CN 107810415; WO 2001 / 087485; EP 3782722; CN 102186992; and JP 6857029; all of which are incorporated by reference herein in their entirety and for all purposes.

[0030] “Solid support” refers to the substrate on which the sample is placed. A microfluidic device is adhered to the solid support, such that the solid support forms the base layer or bottom of the microfluidic device. In embodiments, the solid support comprises one layer. In embodiments, the solid support comprises from 2 to about 6 layers. In embodiments, the solid support comprises 2 layers. In embodiments, the solid support is a “coverslip” A “coverslip” is a thin, flat, transparent substrate. In embodiments, a “coverslip” has a thickness from about 0.13 mm to about 0.2 mm, or from about 0.13 mm to about 0.19 mm, or from about 0.13 mm to about 0.18 mm, or from about 0.13 mm to about 0.17 mm. In embodiments, the coverslip is glass, alternatively referred to as a “glass coverslip” or “glass slide.” In embodiments, the solid supportcomprises a coverslip and a glass layer. In embodiments, the solid support comprises a coverslip and a glass layer, wherein the glass layer has a thickness that is equal to or greater than the thickness of the coverslip. In embodiments, the solid support comprises a coverslip and a glass layer, wherein the glass layer has a thickness that is greater than the thickness of the coverslip. In embodiments, the solid support is a coverslip made from soda lime glass, float glass, borosilicate, quartz, UV fused silica, sapphire, or calcium fluoride. In embodiments, the solid support is glass. In embodiments, the solid support is soda lime glass. In embodiments, the solid support is quartz. In embodiments, the solid support is float glass. In embodiments, the solid support is borosilicate. In embodiments, the solid support is UV fused silica. In embodiments, the solid support is sapphire. In embodiments, the solid support is calcium fluoride.

[0031] “Polydimethylsiloxane” or “PDMS” refers to a silicone polymer with chemical formula CH3[Si(CH3)2O]nSi(CH3)3, where n is the number of repeating monomer units, i.e., [Si(CH3)2O]. PDMS can be prepared according to methods known in the art. In embodiments, PDMS is prepared by weighing out a crosslinker (Part B) component and a base (Part A) component in specific ratio, such as A:B in a ratio of about 20 to about 1. The components are mixed and applied accordingly. In embodiments, PDMS is the compound identified by CAS Registry No.9016-00-6. In embodiments, MOMENTIVE® RTV615 Potting and Encapsulating Compound (Momentive Performance Materials LLC, Waterford NY) is used.

[0032] “Microarray format” is used in accordance with its plain and ordinary meaning in the art and generally refers to a format in which individual spatial areas are arranged on a solid support. In embodiments, the microarray format may include hundreds or thousands of individual spatial areas (e.g. spots) in defined positions. A microarray format is used in laboratory techniques for hybridizing a nucleic acid sample to a large set of oligonucleotides attached to a solid surface for gene expression analysis or genetic variation analysis. During preparation, the sample is applied to the solid surface in predetermined and precise locations so that reactions can be monitored. In embodiments, a microarray format involves the sample deposited onto the solid surface in a number of rows by a number of columns.

[0033] “Individual spatial areas” refers to the area of a solid surface which a sample is applied. The sample, such as suspended in a solution, is applied at known locations on the solid support (e.g., a glass slide). A set volume, often in the range of 375 picoliters to 425 picoliters, is deposited on the glass slide with sufficient distance between adjacent sample volumes to prevent unwanted mixing or contamination. The area of the solid support (e.g., glass slide) under a single volumetric sample is referred to as the individual spatial area.

[0034] “Layer” refers to the individual subcomponents of a microfluidic device that are separately formed then aligned with one another and bonded to form a microfluidic device. Each layer can be designed and formed to perform a unique function within the overall device, for example, a flow layer may contain channels for the sample and a control layer can contain channels for controlling the pressure and valves within the microfluidic device. A microfluidic device may contain one, two, three, or more layers that are aligned and baked to permanently fix together. Each layer can be formed from PDMS with methods that are further described herein.

[0035] “Flow channel” refers to a microfluidic channel through which a solution can flow. The dimensions of flow channels can vary widely but typically include at least one cross- sectional dimension (e.g., height, width, or diameter) less than 1 mm, or less than 0.5 mm, or less than 0.3 mm. Flow channels often have at least one cross-sectional dimension in the range of about 0.05 to about 1000 microns, or from about 0.2 microns to about 500 microns, or from about 10 microns to about 250 microns. The channel may have any suitable cross-sectional shape that allows for fluid transport, for example, a square channel, a circular channel, a rounded channel, a rectangular channel, etc.

[0036] “Avidin” or “avidin protein” refers to biotin-binding proteins and includes without limitation, neutralite avidin, streptavidin, or any similar high-affinity biotin-binding proteins. In embodiments, avidin is the compound identified by CAS registry number 1405-69-2. Similarly, the term “biotin” or “biotin protein” refers to an avidin-binding protein that in its customary sense may also be referred to as the compound identified by CAS registry number 58-85-5. Accordingly, “biotin-avidin interaction” as provided herein refers to the high affinity non- covalent binding of biotin to avidin or equivalents thereof.

[0037] “Reaction chamber” refers to a three-dimensional space and / or volume that provides for contacting of the sample to the solid support (e.g., glass slide). The reaction chamber can have an inlet port for the introduction of the sample to be analyzed. Depending on the reaction, multiple inlet ports are used, that may feed from a variety of storage chambers or from the outside of the chamber. The inlet port may optionally comprise a seal to prevent or reduce the evaporation of the sample or reagents from the reaction chamber.

[0038] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0039] A “fusion protein” refers to a chimeric protein containing two or more separate protein sequences. In embodiments, the fusion protein is recombinantly expressed as a single moiety. In embodiments, the “fusion protein” is an “enzyme fusion protein.” An “enzyme fusion protein” comprises a first protein and a second protein, wherein at least one of the proteins is an enzyme or functional portion thereof (e.g., enzyme domain).

[0040] The term “antibody” is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries. The term “antibody” as referred to herein further includes antibody variants such as single domain antibodies. Thus, in embodiments an antibody includes a single monomeric variable antibody domain. Thus, in embodiments, the antibody, includes a variable light chain (VL) domain or a variable heavy chain (VH) domain. In embodiments, the antibody is a variable light chain (VL) domain or a variable heavy chain (VH) domain. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0041] An “antibody variant” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, affibodies (polypeptides smaller than monoclonal antibodies (e.g., about 6kDA) and capable of binding antigens with high affinity and imitating monoclonal antibodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgGs, scFv, bispecific diabodies, trispecifictriabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies.

[0042] The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.

[0043] “Protease” is used according to its commonly known meaning in the art and refers to an enzyme that hydrolyses peptide bonds. Most proteases are translated as inactive zymogens that contain inhibitory prodomain (referred to herein as a “protease activation sequence”) that must be removed for the protease to become active (referred to herein as an “activated protease”). Proteases that are not synthesized with inhibitory prodomain often require cofactor binding or posttranslational modification. This definition of protease also applies to the protease- part of the terms that include the word “protease” described herein. Proteases are classified on the basis of their catalytic mechanism into the following exemplary groups: glutamic proteases, aspartic proteases, cysteine proteases, serine proteases, metalloproteases, and threonine proteases. In embodiments, the protease is a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease. In embodiments, the protease is a glutamic protease. In embodiments, the protease is an aspartic protease. In embodiments, the protease is a cysteine protease. In embodiments, the protease domain is a serine protease. In embodiments, the protease is a metalloprotease. In embodiments, the protease is a threonine protease. In embodiments, the protease is an exopeptidase (an enzyme that hydrolyses peptide bonds at the terminus of a protein) or an endopeptidase (an enzyme that hydrolyses peptide bonds with a protein). In embodiments, the protease is an exopeptidase. In embodiments, the protease is an endopeptidase.

[0044] The term “zymogen” or “inactive protease” or “inactive protease domain” or “inactive protease fusion protein” is used according to its commonly known meaning in the art and refersto a protease comprising a protease activation sequence (inhibitory prodomain) that renders theprotease unable to perform its intended function. For purposes of this disclosure, the terms “protease,” “protease domain,” and “protease fusion protein” refer to “inactive protease,” “inactive protease domain,” “inactive protease fusion protein,” respectively. A protease that does not contain the protease activation sequence is specifically referred to as an “activated protease,” “activated protease domain,” and “activated protease fusion protein,” respectively.

[0045] “Activating protease” is used according to its commonly known meaning in the art andrefers to a protease that cleaves the protease activation sequence of the inactive protease, therebyremoving the protease activation sequence to produce an activated protease. An activating protease is “orthogonal” to the inactive protease (zymogen). In other words, the activating protease will only interact with (i.e., cleave) the inhibitory prodomain of the zymogen and will not interfere with other components of the zymogen, such that the activated protease will retain all functional activity after release of the inhibitory prodomain. Activating proteases are well known in the art. In embodiments, the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease. In embodiments, the activating protease is a tobacco etch virus protease. In embodiments, the activating protease is an enterokinase. In embodiments, the activating protease is a SARS-CoV-23CL protease.

[0046] “Tobacco etch virus protease” as referred to herein includes any of the recombinant or naturally-occurring forms of the tobacco etch virus protease, or variants or homologs thereof, that maintain tobacco etch virus protease activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to tobacco etch virus protease). In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring tobacco etch virus protease. In embodiments, the tobacco etch virus protease is substantially identical to the protein identified by the UniProt reference number Q0GDU8 or a variant or homolog having substantial identity thereto. Numerous variants and homologs of the tobacco etch virus are known in the art, including tobacco etch virus that are commercially available and described in publications and patents.

[0047] “Enterokinase” as referred to herein includes any of the recombinant or naturally- occurring forms of enterokinase, or variants or homologs thereof, that maintain enterokinase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to enterokinase). In embodiments, the variants or homologs have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, or 150 continuous amino acid portion) compared to a naturally occurring enterokinase. In embodiments, the enterokinase is substantially identical to the protein identified by the UniProt reference number V9QHE7 or a variant or homolog having substantial identity thereto. Numerous variants and homologs of enterokinase are known in the art, including enterokinase that are commercially available and described in publications and patents.

[0048] “SARS-CoV-23CL protease” as referred to herein includes any of the recombinant or naturally-occurring forms of SARS-CoV-23CL protease, or variants or homologs thereof, that maintain SARS-CoV-23CL protease activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to SARS-CoV-23CL protease). In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, or 150 continuous amino acid portion) compared to a naturally occurring SARS-CoV-23CL protease. In embodiments, the SARS-CoV-23CL protease is the SARS-CoV main protease (Mpro), which is substantially identical to the protein identified by NCBI Reference Sequence: YP_009725301.1 or a variant or homolog having substantial identity thereto. Numerous variants and homologs of enterokinase are known in the art, including enterokinase that are commercially available and described in publications and patents.

[0049] “Protease activation sequence” is used according to its commonly known meaning inthe art and refers to an inhibitory prodomain bound to a protease that prevents the protease fromhaving any functional activity. It is well known in the art that the amino acid sequence of the protease activation sequence will be dependent upon the activating protease selected for use in the methods described herein. Protease activation sequences are well known in the art and described throughout the literature. For example, when the activating protease is a tobacco etch virus protease, the protease activation sequence is EX1LYX2Q (SEQ ID NO:1) where X1is any residue and X2is any large or medium residue. In embodiments, the protease activation sequence is SEQ ID NO:2. Prior to cleavage by the activating protease, SEQ ID NO:1 and SEQ ID NO:2 further comprise an additional amino acid immediately after the cleavage site of glutamine (Q), wherein the additional amino acid is G, S, A, M, N, H, Y, Q, F, C, K, W, T, L, E, R, D, V, or I. In embodiments, the additional amino acid is G, S, A, M, N, H, Y, Q, F, C, K, W, T, L, or E. In embodiments, the additional amino acid is G, S, A, M, N, H, Y, Q, or F. In embodiments, the further amino acid is G, S, A, M, or N. In embodiments, the additional aminoacid is G or S. In embodiments, the additional amino acid is G. In embodiments, the additional amino acid is S. The additional amino acid can impact the efficiency and extent of cleavage of the protease activation sequence from the protease domain. As another example, when the activating protease is enterokinase, the protease activation sequence is SEQ ID NO:3.

[0050] “Protease fusion protein” refers to a fusion protein comprising a protease domain, a protease activation sequence, and a fluorescent protein tag. In embodiments, a protease fusion protein further comprises a detectable N-terminal tag.

[0051] “Activated protease fusion protein” refers to a fusion protein comprising a protease domain and a fluorescent protein tag. An activated protease fusion protein does not comprise a protease activation sequence or a detectable N-terminal tag.

[0052] “Protease domain” refers to the amino acid sequence of the protease in a protease fusion protein. In embodiments, the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease. In embodiments, the protease domain comprises a glutamic protease. In embodiments, the protease domain comprises an aspartic protease. In embodiments, the protease domain comprises a cysteine protease. In embodiments, the protease domain comprises a serine protease. In embodiments, the protease domain comprises a metalloprotease. In embodiments, the protease domain comprises a threonine protease. In embodiments, the protease domain comprises an exopeptidase or an endopeptidase. In embodiments, the protease domain comprises an exopeptidase. In embodiments, the protease domain comprises an endopeptidase.

[0053] “Activated protease domain” refers to the amino acid sequence of the protease in a protease fusion protein when a protease activation sequence is not bound to the protease.

[0054] “Detectable N-terminal tag” or “epitope tag” is used according to its commonly knownmeaning in the art and refers to a peptide that is fused to a protein in order to identify the proteinusing commercially available antibodies or assays. In embodiments, the detectable N-terminal tag is an ALFA-tag, glutathione S-transferase (GST), Myc, FLAG-tag, 3X FLAG-tag, HA-tag, polyhistidine-tag (His-tag), or V5-tag. In embodiments, the detectable N-terminal tag is an ALFA-tag. In embodiments, the detectable N-terminal tag is GST. In embodiments, the detectable N-terminal tag is Myc. In embodiments, the detectable N-terminal tag is FLAG-tag. In embodiments, the detectable N-terminal tag is an 3X FLAG-tag. In embodiments, the detectable N-terminal tag is an HA-tag. In embodiments, the detectable N-terminal tag is an His- tag. In embodiments, the detectable N-terminal tag is an V5.

[0055] “Detectable substrate” refers to a compound designed to directly or indirectly release a detectable signal in response to specific biochemical interactions to assess the activity of proteases. The detectable substrate can be any compound that directly or indirectly emits a detectable signal, such as a fluorescent signal, spectrophotometric signal, chromogenic signal, bioluminescent signal, near-infrared signal, colorimetric, radiometric, or a signal detectable by surface-enhanced Raman spectroscopy.

[0056] “Detectable signal” refers to the signal emitted by a detectable substrate.

[0057] “Fluorogenic substrate” is used according to its commonly known meaning in the art and refers to a molecule that releases a fluorescent signal when it undergoes a specific biochemical interaction. These substrates are generally made up of two parts: an enzyme-labile moiety and a fluorogenic moiety. When an enzyme acts on the substrate, the fluorogenic moiety is cleaved off, which produces a fluorescent signal. that can be detected and quantified. In embodiments, the fluorogenic substrate has an excitation wavelength of about 300 nm or more. In embodiments, the fluorogenic substrate has an excitation wavelength of about 340 nm or more. In embodiments, the fluorogenic substrate is a fluorescence resonance energy transfer (FRET) substrate.

[0058] “Fluorescent signal” is used according to its commonly known meaning in the art andrefers to the light emitted by a molecule after it absorbs light or other electromagnetic radiationand returns to its ground state.

[0059] “Bound” is used according to its commonly known meaning in the art and refers to the covalent attachment between two atoms or two moieties. The covalent attachment can be direct, i.e., via a covalent bond, or indirect, i.e., via a peptide linker.

[0060] “Peptide linker” is used according to its commonly known meaning in the art and refers to a short peptide sequence that connects different protein or different protein domains. In embodiments, a peptide linker is stable and not susceptible to protease cleavage. In embodiments, the peptide linker comprises from about 1 to about 50 amino acid residues. In embodiments, the peptide linker comprises from about 1 to about 40 amino acid residues. In embodiments, the peptide linker comprises from about 1 to about 30 amino acid residues. In embodiments, the peptide linker comprises from about 1 to about 25 amino acid residues. In embodiments, the peptide linker comprises from about 1 to about 20 amino acid residues. In embodiments, the peptide linker comprises from about 1 to about 15 amino acid residues. In embodiments, the peptide linker comprises from about 1 to about 10 amino acid residues. Inembodiments, the peptide linker comprises from about 1 to about 5 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 50 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 45 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 40 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 35 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 30 amino acid residues In embodiments, the peptide linker comprises from about 5 to about 25 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 20 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 15 amino acid residues. In embodiments, the peptide linker comprises from about 5 to about 10 amino acid residues.

[0061] In embodiments, the peptide linker is a “Gly-Ser linker” which refers to a peptide that includes at least one serine and at least one glycine. In embodiments, the peptide linker includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7) glycine amino acid residues. In embodiments, the peptide linker has a length of less than 20 (e.g., 19, 18, 17, 16, etc.) amino acid residues. In embodiments, the Gly-Ser linker is GlyzSer(Gly4Ser)xGly, wherein y is in an integer from 1 to 3 and x is an integer from 1 to 10. In embodiments, y is 1. In embodiments, y is 2. In embodiments, y is 3. In embodiments, y is 3 and x is 1. In embodiments, the Gly-Ser linker is (Gly4Ser)x, wherein x is an integer from 1 to 10. In embodiments, x is an integer from 1 to 9. In embodiments, x is an integer from 1 to 8. In embodiments, x is an integer from 1 to 7. In embodiments, x is an integer from 1 to 6. In embodiments, x is an integer from 1 to 5. In embodiments, x is an integer from 1 to 4. In embodiments, x is an integer from 1 to 3. In embodiments, x is an integer from 1 to 2. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10. In embodiments, the Gly-Ser linker is preceded by (GlyzSer)- and / or followed by –(Glyz), wherein z is in an integer from 1 to 3. In embodiments, z is 1. In embodiments, z is 2. In embodiments, z is 3.

[0062] A “detectable agent” or “detectable moiety” is a composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIOnanoparticle aggregates, superparamagnetic iron oxide (“SPIO”) nanoparticles, SPIO nanoparticle aggregates, monocrystalline iron oxide nanoparticles, monochrystalline iron oxide, Gadolinium, radioisotopes, radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and / or polymers; microbubble gas core including air, heavy gases, perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.), iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. A detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.

[0063] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. antibodies and antigens) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated; however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. In embodiments, “contacting” is reacting.

[0064] “About” refers to a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the terms “about” mean within a standard deviation using measurements generally acceptable in the art. In embodiments, “about” means a range extending to + / - 10% of the specified value. In embodiments, “about” means a range extending to + / - 5% of the specified value. In embodiments, “about” means the specified value.

[0065] The terms “a” or “an,” as used in herein means one or more.

[0066] Apparatus

[0067] Provided herein are devices for improved high throughput microfluidic platforms capable of assessing protease activity. For example, a two-layer microfluidic device with integrated pneumatic valves is optimized to increase the number of chambers simultaneously measuring catalytic activities, improve device durability and simplify functionalization withalternative glass coating techniques, and enhance the signal to noise ratio.

[0068] In embodiments, a solid support is provided. A first surface of the solid support is coated with a PDMS layer. A plurality of individual spatial areas in a microarray format is deposited on the PDMS layer on the first surface of the solid support. Each of the plurality of individual spatial areas comprises a plurality of plasmids encoding a protease fusion protein.

[0069] In embodiments, the PDMS layer on the first surface of the solid support has a thickness of about 1 µm to about 120 µm. In embodiments, the PDMS layer on the first surface of the solid support has a thickness of about 1 µm to about 110 µm. In embodiments, the PDMS layer on the first surface of the solid support has a thickness of about 1 µm to about 100 µm. In embodiments, the PDMS layer has a thickness of about 1 µm to about 100 µm. In embodiments, the PDMS layer has a thickness of about 10 µm to about 90 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 80 µm. In embodiments, the PDMS layer has a thickness of about 25 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 75 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 70 µm. In embodiments, the PDMS layer with a thickness of about 20 µm to about 65 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 60 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 55 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 50 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 45 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to 40 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 35 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 30 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 25 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 75 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 70 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 65 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 60 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 55 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 50 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 45 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 40 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 35 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 30 µm. In embodiments, the PDMS layer has a thickness of about 20 µm to about 25 µm.

[0070] In embodiments, the PDMS polymer has a ratio of base component (part A) tocrosslinker component (part B) from about 5:1 to about 50:1. In embodiments, the PDMS polymer has a ratio of part A to part B from about 10:1 to about 40:1. In embodiments, the PDMS polymer has a ratio of part A to part B from about 10:1 to about 30:1. In embodiments, the PDMS polymer has a ratio of part A to part B from about 10:1 to about 20:1. In embodiments, the PDMS polymer has a ratio of part A to part B from about 15:1 to about 25:1. In embodiments, the PDMS polymer has a ratio of part A to part B from about 15:1 to about 20:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 20:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 10:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 19:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 18:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 17:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 16:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 15:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 14:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 13:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 12:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 11:1. In embodiments, the PDMS polymer has a ratio of part A to part B of about 10:1.

[0071] In embodiments, each of the plurality of individual spatial areas deposited onto the solid support (e.g., glass slide) has a volume between 375 picoliter and 425 picoliter. It can be appreciated that larger volumes of spatial areas can be implemented, such as volumes up to 850 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 250 picoliter to about 850 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 300 picoliter to about 800 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 300 picoliter to about 700 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 300 picoliter to about 600 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 300 picoliter to about 500 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 350 picoliter to about 450 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 800 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 750 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 700 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 650 picoliter. In embodiments, each of the plurality of individual spatial areashas a volume of about 375 picoliter to about 600 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 550 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 500 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 450 picoliter. In embodiments, each of the plurality of individual spatial areas has a volume of about 375 picoliter to about 400 picoliter.

[0072] In embodiments, the solid support (e.g., glass slide) arrayed with individual spatial areas forms part of a microfluidic system when combined with a microfluidic device. In embodiments, the microfluidic device is sealed to a first surface (top) of the solid support (e.g., glass slide). The microfluidic device has a first end and an opposing second end, a plurality of sample chambers (e.g., at least 2, 5, 10, 100, 500, etc.) configured to enclose the plurality of individual spatial areas on the solid support (e.g., glass slide), a plurality of reaction chambers (e.g., at least 2, 5, 10, 100, 500, etc.) in fluid communication with the plurality of sample chambers by a network of flow channels therebetween, a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers, a plurality of control channels configured to open and close the plurality of valves, at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels, and an outlet disposed at the second end in fluid communication with the network of flow channels.

[0073] In embodiments, the microfluidic device has a first layer with the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves. The first layer is bound to a second layer having the plurality of control channels. One or both layers of the microfluidic device are made of PDMS. The fluidic inlet is a plurality of control inlets and a plurality of solution inlets. The control inlets are in fluid communication with the plurality of control channels such that pressure is controlled within the device to open and close the plurality of valves. The plurality of solution inlets is in fluid communication with at least one reagent. A list of some of the reagents used with the device are listed in the following “Examples” section. For example, phosphate buffer solution, bovine serum albumin, and water.

[0074] In embodiments, the total number of chambers, the sample chambers and the reaction chambers is 1792. Devices with more or less chambers can be appreciated based on the application of the device, and the total number of chambers, the sample chambers and the reaction chambers, could be between 1500 to 2000. Each of the sample chambers encloses one individual spatial area of the individual spatial areas on the solid support (e.g., glass slide) whenthe microfluidic device is aligned with the solid support (e.g., glass slide). The plurality of valves can include any combination of one or more of sandwich valves, button valves, and neck valves. As fluid flows through the device from the fluid inlet, fluid flows out of the device at the outlet which transports waste out of the microfluidic system.

[0075] In embodiments, a solid support has a length between 45 mm and 55 mm, a width between 60 mm and 70 mm, and a thickness (depth) from about 0.01 mm to about 1 mm and a plurality of individual spatial areas can be deposited in a microarray format on a first surface of the solid support (e.g., glass slide). Like previously described, an avidin protein is bound to the first surface of the solid support (e.g., glass slide) and bound to a biotinylated anti-tag protein antibody. Each of the plurality of individual spatial areas includes the avidin protein and a plurality of plasmids encoding an enzyme fusion protein. The enzyme fusion protein may include an enzyme domain and a protein tag domain, and the protein tag domain may be capable of binding to the biotinylated anti-tag protein antibody.

[0076] Solid supports with different dimensions may be used. In embodiments, the solid support has a length between about 30 mm and about 100 mm. In embodiments, the solid support has a length between about 45 mm and about 70 mm. In embodiments, the solid support has a length between about 45 mm and about 65 mm. In embodiments, the solid support has a length between about 45 mm and about 60 mm. In embodiments, the solid support has a length between about 45 mm and 55 mm. In embodiments, the solid support has a length between 45 mm and about 70 mm. In embodiments, the solid support has a length between 45 mm and about 65 mm. In embodiments, the solid support has a length between 45 mm and about 60 mm. In embodiments, the solid support has a length between 45 mm and about 55 mm.

[0077] In embodiments, the solid support has a width between about 40 mm and about 100 mm. In embodiments, the solid support has a width between about 60 mm and about 85 mm. In embodiments, the solid support has a length between about 60 mm and about 80 mm. In embodiments, the solid support has a length between about 60 mm and about 75 mm. In embodiments, the solid support has a length between about 60 mm and about 70 mm. In embodiments, the solid support has a width between 60 mm and about 85 mm. In embodiments, the solid support has a length between 60 mm and about 80 mm. In embodiments, the solid support has a length between 60 mm and about 75 mm. In embodiments, the solid support has a length between 60 mm and about 70 mm.

[0078] In embodiments, the solid support has a thickness from about 25 µm to about 1.5 mm. In embodiments, the solid support has a thickness from about 50 µm to about 1.0 mm. Inembodiments, the solid support has a thickness from about 50 µm to about 500 µm. In embodiments, the solid support has a thickness from about 50 µm to about 400 µm. In embodiments, the solid support has a thickness from about 50 µm to about 300 µm. In embodiments, the solid support has a thickness from about 80 µm to about 250 µm. In embodiments, the solid support has a thickness from about 80 µm to about 190 µm. In embodiments, the solid support has a thickness from about 80 µm to about 170 µm. In embodiments, the solid support has a thickness from about 100 µm to about 250 µm. In embodiments, the solid support has a thickness from about 100 µm to about 240 µm. In embodiments, the solid support has a thickness from about 100 µm to about 190 µm. In embodiments, the solid support has a thickness from about 100 µm to about 170 µm. In embodiments, the solid support has a thickness from about 100 µm to about 150 µm. In embodiments, the solid support has a thickness from about 100 µm to about 130 µm. In embodiments, the solid support has a thickness from about 130 µm to about 190 µm. In embodiments, the solid support has a thickness from about 130 µm to about 170 µm. In embodiments, the solid support has a thickness from about 150 µm to about 250 µm. In embodiments, the solid support has a thickness from about 150 µm to about 190 µm. In embodiments, the solid support has a thickness from about 150 µm to about 170 µm. In embodiments, the solid support has a thickness from about 160 µm to about 250 µm. In embodiments, the solid support has a thickness from about 170 µm to about 250 µm. In embodiments, the solid support has a thickness from about 170 µm to about 190 µm. In embodiments, the solid support has a thickness from about 85 µm to about 130 µm. In embodiments, the solid support has a thickness from about 130 µm to about 160 µm. In embodiments, the solid support has a thickness from about 190 µm to about 230 µm. In embodiments, the solid support has a thickness of about 100 µm. In embodiments, the solid support has a thickness of about 130 µm. In embodiments, the solid support has a thickness of about 150 µm. In embodiments, the solid support has a thickness of about 220 µm.

[0079] In embodiments, the solid support has a thickness from about 150 µm to about 200 µm. In embodiments, the solid support has a thickness from about 160 µm to about 190 µm. In embodiments, the solid support has a thickness from about 160 µm to about 180 µm. In embodiments, the solid support has a thickness from about 165 µm to about 175 µm. In embodiments, the solid support has a thickness of about 170 µm. In embodiments, the solid support is a glass coverslip.

[0080] In embodiments, the solid support is about 50 mm in length and about 64 mm in widthand about 0.17 mm in thickness. In embodiments, the solid support is a CLARITEX® Supa Mega Coverslip (No.1.550 x 64 mm).

[0081] Solid Support

[0082] Provided herein is a solid support comprising a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a protease activation sequence, and (iii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to the first surface of the solid support. In embodiments, the solid support is glass. In embodiments, the solid support is quartz. In embodiments, a first surface of the solid support comprises a layer of polydimethylsiloxane. In embodiments, the polydimethylsiloxane has a ratio of base component to crosslinker component from about 10:1 to about 30:1. In embodiments, the polydimethylsiloxane has a ratio of base component to crosslinker component of about 20:1. In embodiments, the layer of polydimethylsiloxane has a thickness from about 1 uM to about 120 uM. In embodiments, the layer of polydimethylsiloxane has a thickness from about 20 uM to about 80 uM. In embodiments, the solid support has a thickness from about 10 µm to about 1 mm. In embodiments, the solid support has a thickness from about 100 µm to about 240 µm. In embodiments, the solid support has a thickness of about 170 µm. In embodiments, the solid support has a length from about 30 mm to about 100 mm and a width from about 40 mm to about 100 mm. In embodiments, the solid support has a length from about 45 mm to about 55 mm and a width from about 60 mm to about 70 mm. In embodiments, the protease fusion protein further comprises a detectable N-terminal tag, wherein the detectable N-terminal tag is bound to the protease activation sequence. In embodiments, the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease. In embodiments, the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain. In embodiments, the protease activation sequence is SEQ ID NO:1 or SEQ ID NO:2 when the activating protease is a tobacco etch virus protease. In embodiments, the protease activation sequence is SEQ ID NO:3 when the activating protease is an enterokinase. In embodiments, the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease. In embodiments, the protease domain comprises an exopeptidase or an endopeptidase. In embodiments, the protease fusion protein further comprises a detectable N-terminal tag, wherein the detectable N-terminal tag is bound to the protease activation sequence. In embodiments, the detectable N-terminal tag is an ALFA-tag, glutathione S-transferase, Myc,FLAG-tag, 3X FLAG-tag, HA-tag, polyhistidine-tag, or V5-tag-tag. In embodiments, the detectable substrate is a fluorogenic substrate and the detectable signal is a fluorescent signal. In embodiments, the detectable substrate is a fluorogenic substrate that has an excitation wavelength of about 340 nm or more. In embodiments, the detectable substrate is a FRET substrate. In embodiments, the detectable substrate is a near infrared substrate and the detectable signal is a near infrared signal. In embodiments, the detectable signal is a signal detectable by surface-enhanced Raman spectroscopy. In embodiments, the fluorescent protein tag is bound to the C-terminus of the protease domain. In embodiments, the fluorescent protein tag is bound to N-terminus of the protease domain. In embodiments, the fluorescent protein tag is green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein. In embodiments, the fluorescent protein tag is enhanced green fluorescent protein. In embodiments, the fluorescent protein tag is bound to the protease domain via a peptide linker. In embodiments, the peptide linker is a Gly- Ser linking group. In embodiments, the Gly-Ser linking group is (Gly4Ser)x, wherein x is an integer from 1 to 10. In embodiments, the fluorescent protein tag is bound to the solid support via an anti-fluorescent protein tag antibody. In embodiments, the fluorescent protein tag is bound to an anti-fluorescent protein tag antibody; the anti-fluorescent protein tag antibody is bound to a neutralite avidin protein; and the neutralite avid protein is bound to the solid support. In embodiments, the anti-fluorescent protein tag antibody is an anti-fluorescent protein tag variable heavy domain of heavy chain.

[0083] Microfluidic System

[0084] Provided herein is a microfluidic system comprising a microfluidic device and the solid support as described herein, including embodiments thereof. In embodiments, the microfluidic device is adjacent the first surface of the solid support. In embodiments, the microfluidic device is adhered to the polydimethylsiloxane on the first surface of the solid support. In embodiments, wherein the microfluidic device comprises a plurality of individual spatial areas in a microarray format. In embodiments, the plurality of individual spatial areas have a volume from about 300 picolitres to about 500 picolitres. In embodiments, the microfluidic device comprises: (a) a first end opposite a second end; (b) a top end and a bottom end, wherein the bottom end is adhered to the solid support; (c) a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the solid support; (d) a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween; (e) a plurality of valves configured to control fluidmovement between the plurality of sample chambers and the plurality of reaction chambers; (f) a plurality of control channels configured to open and close the plurality of valves; (g) at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and (h) an outlet disposed at the second end in fluid communication with the network of flow channels. In embodiments, the microfluidic device comprises (i) a first layer comprising the plurality of control channels, and (ii) a second layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves; wherein the first layer is adhered to the second layer and the second layer is adhered to the solid support. In embodiments, the first layer comprises polydimethylsiloxane and the second layer comprises polydimethylsiloxane. In embodiments, the polydimethylsiloxane in the second layer is crosslinked with the layer of polydimethylsiloxane on the solid support. In embodiments, the at least one fluidic inlet comprises a plurality of control inlets and a plurality of solution inlets. In embodiments, the plurality of control inlets at the first end of the microfluidic device is in fluid communication with the plurality of control channels. In embodiments, the plurality of solution inlets is in fluid communication with at least one reagent. In embodiments, the microfluidic device comprises 1792 chambers, wherein the total number of chambers comprises the plurality of sample chambers plus the plurality of reaction chambers. In embodiments, each sample chamber of the plurality of sample chambers encloses one individual spatial area of the plurality of individual spatial areas on the solid support. In embodiments, the plurality of valves comprises sandwich valves, button valves, and neck valves. In embodiments, the outlet is configured to transport waste out of the microfluidic system. In embodiments, the microfluidic device further comprises an activating protease. In embodiments, the microfluidic device further comprises an activating protease selected from the group consisting of a tobacco etch virus protease, an enterokinase, and a SARS-CoV-23CL protease.

[0085] Methods

[0086] Provided herein is a method for assessing protease activity in a microfluidic system comprising: (a) reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a protease activation sequence, wherein the protease activation sequence is bound to the protease domain, and (ii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a first surface of a solid support; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; (b) reacting the activated protease fusion protein with adetectable substrate, thereby generating a detectable signal; and (c) detecting the detectable signal, thereby detecting protease activity in the microfluidic system. In embodiments, the protease fusion protein further comprises a detectable N-terminal tag, wherein the detectable N- terminal tag is bound to the protease activation sequence. In embodiments, the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease. In embodiments, the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain. In embodiments, the protease activation sequence is SEQ ID NO:1 or SEQ ID NO:2 when the activating protease is a tobacco etch virus protease. In embodiments, the protease activation sequence is SEQ ID NO:3 when the activating protease is an enterokinase. In embodiments, the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease. In embodiments, the protease domain comprises an exopeptidase or an endopeptidase. In embodiments, the detectable N-terminal tag is an ALFA-tag, glutathione S-transferase, Myc, FLAG-tag, 3X FLAG-tag, HA-tag, polyhistidine-tag, or V5-tag-tag. In embodiments, the detectable substrate is a fluorogenic substrate and the detectable signal is a fluorescent signal. In embodiments, the detectable substrate is a fluorogenic substrate that has an excitation wavelength of about 340 nm or more. In embodiments, the detectable substrate is a FRET substrate. In embodiments, the detectable substrate is a near infrared substrate and the detectable signal is a near infrared signal. In embodiments, the detectable signal is a signal detectable by surface-enhanced Raman spectroscopy. In embodiments, the fluorescent protein tag is bound to the C-terminus of the protease domain. In embodiments, the fluorescent protein tag is bound to N-terminus of the protease domain. In embodiments, the fluorescent protein tag is green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein. In embodiments, the fluorescent protein tag is enhanced green fluorescent protein. In embodiments, the fluorescent protein tag is bound to the protease domain via a peptide linker. In embodiments, the peptide linker is a Gly- Ser linking group. In embodiments, the Gly-Ser linking group is (Gly4Ser)x, wherein x is an integer from 1 to 10. In embodiments, the fluorescent protein tag is bound to the solid support via an anti-fluorescent protein tag antibody. In embodiments, the fluorescent protein tag is bound to an anti-fluorescent protein tag antibody; the anti-fluorescent protein tag antibody is bound to a neutralite avidin protein; and the neutralite avid protein is bound to the solid support. In embodiments, the anti-fluorescent protein tag antibody is an anti-fluorescent protein tag variable heavy domain of heavy chain. In embodiments, the methods further comprising, prior to step (a), expressing a plasmid to produce the protease fusion protein.

[0087] Provided herein is a method for assessing protease activity in a microfluidic system comprising: (a) reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a protease activation sequence, wherein the protease activation sequence is bound to the protease domain, (iii) a detectable N- terminal tag, wherein the detectable N-terminal tag is bound to the protease activation sequence; and (iv) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a first surface of a solid support; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; (b) reacting the activated protease fusion protein with a fluorogenic substrate, thereby generating a fluorescent signal; and (c) detecting the fluorescent signal, thereby detecting protease activity in the microfluidic system. In embodiments of the methods described herein the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease. In embodiments, the activating protease is a tobacco etch virus protease. In embodiments, the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain. In embodiments, the protease activation sequence is SEQ ID NO:1 or SEQ ID NO:2 when the activating protease is a tobacco etch virus protease. In embodiments, the protease activation sequence is SEQ ID NO:3 when the activating protease is an enterokinase. In embodiments, the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease. In embodiments, the protease domain comprises an exopeptidase or an endopeptidase. In embodiments, the detectable N-terminal tag is an ALFA-tag, glutathione S-transferase, Myc, FLAG-tag, 3X FLAG-tag, HA-tag, polyhistidine-tag, or V5-tag-tag. In embodiments, the fluorogenic substrate has an excitation wavelength of about 340 nm or more. In embodiments, the detectable substrate is a FRET substrate. In embodiments, the fluorescent protein tag is bound to the C-terminus of the protease domain. In embodiments, the fluorescent protein tag is bound to N-terminus of the protease domain. In embodiments, the fluorescent protein tag is green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein. In embodiments, the fluorescent protein tag is enhanced green fluorescent protein. In embodiments, the fluorescent protein tag is bound to the protease domain via a peptide linker. In embodiments, the peptide linker is a Gly-Ser linking group. In embodiments, the Gly-Ser linking group is (Gly4Ser)x, wherein x is an integer from 1 to 10. In embodiments, the fluorescent protein tag is bound to the solid support via an anti-fluorescent protein tag antibody. In embodiments, the fluorescent protein tag is bound to an anti-fluorescentprotein tag antibody; the anti-fluorescent protein tag antibody is bound to a neutralite avidin protein; and the neutralite avid protein is bound to the solid support. In embodiments, the anti- fluorescent protein tag antibody is an anti-fluorescent protein tag variable heavy domain of heavy chain. In embodiments, the methods further comprising, prior to step (a), expressing a plasmid to produce the protease fusion protein.

[0088] Provided herein is a method for assessing protease activity in a microfluidic system comprising: (a) reacting an activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; wherein the activated protease fusion protein comprises a protease domain bound to a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support; and (b) detecting the detectable signal, thereby detecting protease activity in the microfluidic system. In embodiments, the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease. In embodiments, the protease domain comprises an exopeptidase or an endopeptidase. In embodiments, the detectable substrate is a fluorogenic substrate and the detectable signal is a fluorescent signal. In embodiments, the detectable substrate is a fluorogenic substrate that has an excitation wavelength of about 340 nm or more. In embodiments, the detectable substrate is a FRET substrate. In embodiments, the detectable substrate is a near infrared substrate and the detectable signal is a near infrared signal. In embodiments, the detectable signal is a signal detectable by surface-enhanced Raman spectroscopy. In embodiments, the fluorescent protein tag is bound to the C-terminus of the protease domain. In embodiments, the fluorescent protein tag is bound to N-terminus of the protease domain. In embodiments, the fluorescent protein tag is green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein. In embodiments, the fluorescent protein tag is enhanced green fluorescent protein. In embodiments, the fluorescent protein tag is bound to the protease domain via a peptide linker. In embodiments, the peptide linker is a Gly-Ser linking group. In embodiments, the Gly-Ser linking group is (Gly4Ser)x, wherein x is an integer from 1 to 10. In embodiments, the fluorescent protein tag is bound to the solid support via an anti-fluorescent protein tag antibody. In embodiments, the fluorescent protein tag is bound to an anti-fluorescent protein tag antibody; the anti-fluorescent protein tag antibody is bound to a neutralite avidin protein; and the neutralite avid protein is bound to the solid support. In embodiments, the anti- fluorescent protein tag antibody is an anti-fluorescent protein tag variable heavy domain of heavy chain.

[0089] Provided herein is a method for assessing protease activity in a microfluidic system comprising: (a) reacting an activated protease fusion protein with a fluorogenic substrate, thereby generating a fluorescent signal; wherein the activated protease fusion protein comprises a protease domain bound to a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support; and (b) detecting the fluorescent signal, thereby detecting protease activity in the microfluidic system. In embodiments, the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease. In embodiments, the protease domain comprises an exopeptidase or an endopeptidase. In embodiments, the fluorogenic substrate has an excitation wavelength of about 340 nm or more. In embodiments, the detectable substrate is a FRET substrate. In embodiments, the fluorescent protein tag is bound to the C-terminus of the protease domain. In embodiments, the fluorescent protein tag is bound to N-terminus of the protease domain. In embodiments, the fluorescent protein tag is green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein. In embodiments, the fluorescent protein tag is enhanced green fluorescent protein. In embodiments, the fluorescent protein tag is bound to the protease domain via a peptide linker. In embodiments, the peptide linker is a Gly-Ser linking group. In embodiments, the Gly-Ser linking group is (Gly4Ser)x, wherein x is an integer from 1 to 10. In embodiments, the fluorescent protein tag is bound to the solid support via an anti-fluorescent protein tag antibody. In embodiments, the fluorescent protein tag is bound to an anti-fluorescent protein tag antibody; the anti-fluorescent protein tag antibody is bound to a neutralite avidin protein; and the neutralite avid protein is bound to the solid support. In embodiments, the anti- fluorescent protein tag antibody is an anti-fluorescent protein tag variable heavy domain of heavy chain.

[0090] Informal Sequence Listing

[0091] SEQ ID NO:1 is EX1LYX2Q, where X1is any residue and X2is any large or medium residue.

[0092] SEQ ID NO:2 is ENLYFQ

[0093] SEQ ID NO:3 is DDDDK

[0094] SEQ ID NO:4 is SAVLQSGFRK

[0095] SEQ ID NO:5 is ENLYFQSGFRK

[0096] SEQ ID NO:6 is ENLYFASGFRK

[0097] SEQ ID NO:7 is ENLYFA

[0098] SEQ ID NO:8 is LGSAVLQ

[0099] Embodiments G1 to G25.

[0100] Embodiment G1. A solid support comprising a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises: (a) a protease domain, (b) a protease activation sequence, and (c) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to the first surface of the solid support.

[0101] Embodiment G2. The solid support of Embodiment G1, wherein the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain.

[0102] Embodiment G3. The solid support of Embodiment G1 or G2, wherein a first surface of the solid support comprises a layer of polydimethylsiloxane.

[0103] Embodiment G4. The solid support of Embodiment G3, wherein the polydimethylsiloxane has a ratio of base component to crosslinker component from about 10:1 to about 30:1.

[0104] Embodiment G5. A microfluidic system comprising a microfluidic device and the solid support of any one of Embodiments G1 to G4.

[0105] Embodiment G6. The microfluidic system of Embodiment G5, wherein the microfluidic device is adjacent the first surface of the solid support.

[0106] Embodiment G7. The microfluidic system of Embodiment G5 or G6, wherein the microfluidic device is adhered to the polydimethylsiloxane on the first surface of the solid support.

[0107] Embodiment G8. The microfluidic system of any one of Embodiments G5 to G7, wherein the microfluidic device comprises: (a) a first end opposite a second end; (b) a top end and a bottom end, wherein the bottom end is adhered to the solid support; (c) a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the solid support; (d) a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween; (e) a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers; (f) a plurality of control channels configured to open and close the plurality of valves;(g) at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and (h) an outlet disposed at the second end in fluid communication with the network of flow channels.

[0108] Embodiment G9. The microfluidic system of any one of Embodiments G5 to G8, wherein the microfluidic device comprises (a) a first layer comprising the plurality of control channels, and (b) a second layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves; wherein the first layer is adhered to the second layer and the second layer is adhered to the solid support.

[0109] Embodiment G10. The microfluidic system of Embodiment G9, wherein the first layer comprises polydimethylsiloxane and the second layer comprises polydimethylsiloxane.

[0110] Embodiment G11. The microfluidic system of Embodiment G10, wherein the polydimethylsiloxane in the second layer is crosslinked with the layer of polydimethylsiloxane on the solid support.

[0111] Embodiment G12. A method for assessing protease activity in a microfluidic system, the method comprising: (a) reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises: (I) a protease domain, (ii) a protease activation sequence, wherein the protease activation sequence is bound to the protease domain, and (ii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a first surface of a solid support; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; (b) reacting the activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; and (c) detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

[0112] Embodiment G13. The method of Embodiment G12, wherein the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease.

[0113] Embodiment G14. The method of any Embodiment G12 or G13, wherein the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain.

[0114] Embodiment G15. The method of any one of Embodiments G12 to G14, wherein the protease fusion protein further comprises a detectable N-terminal tag, wherein the detectable N- terminal tag is bound to the protease activation sequence.

[0115] Embodiment G16. The method of any one of Embodiments G12 to G15, furthercomprising, prior to step (a), expressing a plasmid to produce the protease fusion protein.

[0116] Embodiment G17. A method for assessing protease activity in a microfluidic system, the method comprising: (a) reacting an activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; wherein the activated protease fusion protein comprises a protease domain bound to a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support; and (b) detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

[0117] Embodiment G18. The method of Embodiment G12 to G17, wherein a first surface of the solid support comprises a layer of polydimethylsiloxane.

[0118] Embodiment G19. The method of Embodiment G18, wherein the polydimethylsiloxane has a ratio of base component to crosslinker component from about 10:1 to about 30:1.

[0119] Embodiment G20. The method of any one of Embodiments G12 to G19, wherein a microfluidic device is adjacent the first surface of the solid support.

[0120] Embodiment G21. The method of Embodiment G20, wherein the microfluidic device is adhered to the polydimethylsiloxane on the first surface of the solid support.

[0121] Embodiment G22. The method of Embodiment G20 or G21, wherein the microfluidic device comprises: (a) a first end opposite a second end; (b) a top end and a bottom end, wherein the bottom end is adhered to the solid support; (c) a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the solid support; (d) a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween; (e) a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers; (f) a plurality of control channels configured to open and close the plurality of valves; (g) at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and (h) an outlet disposed at the second end in fluid communication with the network of flow channels.

[0122] Embodiment G23. The method of any one of Embodiments G20 to G22, wherein the microfluidic device comprises (a) a first layer comprising the plurality of control channels, and (b) a second layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves; wherein the first layer is adhered to the second layer and the second layer is adhered to the solid support.

[0123] Embodiment G24. The method of Embodiment G23, wherein the first layer comprises polydimethylsiloxane and the second layer comprises polydimethylsiloxane.

[0124] Embodiment G25. The method of Embodiment G24, wherein the polydimethylsiloxane in the second layer is crosslinked with the layer of polydimethylsiloxane on the solid support.

[0125] Embodiments 1 to 105

[0126] Embodiment 1. A method for assessing protease activity in a microfluidic system, the method comprising: (a) reacting an activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; wherein the activated protease fusion protein comprises a protease domain bound to a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support; and (b) detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

[0127] Embodiment 2. A method for assessing protease activity in a microfluidic system, the method comprising: (a) reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a protease activation sequence, wherein the protease activation sequence is bound to the protease domain, and (iii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a first surface of a solid support; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; (b) reacting the activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; and (c) detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

[0128] Embodiment 3. The method of Embodiment 2, wherein the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease.

[0129] Embodiment 4. The method of Embodiment 2, wherein the activating protease is a tobacco etch virus protease.

[0130] Embodiment 5. The method of any one of Embodiments 2 to 4, wherein the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain.

[0131] Embodiment 6. The method of any one of Embodiments 2 to 5, wherein the protease activation sequence is SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0132] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease.

[0133] Embodiment 8. The method of any one of Embodiments 1 to 7, wherein the protease domain comprises an exopeptidase or an endopeptidase.

[0134] Embodiment 9. The method of Embodiment 2 to 8, wherein the protease fusion protein further comprises a detectable N-terminal tag, wherein the detectable N-terminal tag is bound to the protease activation sequence.

[0135] Embodiment 10. The method of Embodiment 9, wherein the detectable N-terminal tag is an ALFA-tag, glutathione S-transferase, Myc, FLAG-tag, 3X FLAG-tag, HA-tag, polyhistidine-tag, or V5-tag.

[0136] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the detectable substrate is a fluorogenic substrate and the detectable signal is a fluorescent signal.

[0137] Embodiment 12. The method Embodiment 11, wherein the fluorogenic substrate has an excitation wavelength of about 340 nm or more.

[0138] Embodiment 13. The method of any one of Embodiments 1 to 10, wherein the fluorogenic substrate is a FRET substrate.

[0139] Embodiment 14. The method of any one of Embodiments 1 to 10, wherein the detectable substrate is a near infrared substrate and the detectable signal is a near infrared signal.

[0140] Embodiment 15. The method of any one of Embodiments 1 to 10, wherein the detectable signal is a signal detectable by surface-enhanced Raman spectroscopy.

[0141] Embodiment 16. The method of any one of Embodiments 1 to 15, wherein the fluorescent protein tag is bound to the C-terminus of the protease domain.

[0142] Embodiment 17. The method of any one of Embodiments 1 to 15, wherein the fluorescent protein tag is bound to N-terminus of the protease domain.

[0143] Embodiment 18. The method of any one of Embodiments 1 to 17, wherein the fluorescent protein tag is green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein.

[0144] Embodiment 19. The method of any one of Embodiments 1 to 17, wherein the fluorescent protein tag is enhanced green fluorescent protein.

[0145] Embodiment 20. The method of any one of Embodiments 1 to 19, wherein the fluorescent protein tag is bound to the protease domain via a peptide linker.

[0146] Embodiment 21. The method of Embodiment 20, wherein the peptide linker is a Gly-Ser linking group.

[0147] Embodiment 22. The method of Embodiment 21, wherein the Gly-Ser linking group is (Gly4Ser)x, wherein x is an integer from 1 to 10.

[0148] Embodiment 23. The method of any one of Embodiments 1 to 22, wherein the fluorescent protein tag is bound to the solid support via an anti-fluorescent protein tag antibody.

[0149] Embodiment 24. The method of any one of Embodiments 1 to 22, wherein (i) the fluorescent protein tag is bound to an anti-fluorescent protein tag antibody; (ii) the anti- fluorescent protein tag antibody is bound to a neutralite avidin protein; and (iii) the neutralite avid protein is bound to the solid support.

[0150] Embodiment 25. The method of Embodiment 23 or 24, wherein the anti-fluorescent protein tag antibody is an anti-fluorescent protein tag variable heavy domain of heavy chain.

[0151] Embodiment 26. The method of any one of Embodiments 2 to 25, further comprising, prior to step (a), expressing a plasmid to produce the protease fusion protein.

[0152] Embodiment 27. The method of any one of Embodiments 1 to 26, wherein the solid support is glass.

[0153] Embodiment 28. The method of any one of Embodiments 1 to 26, wherein the solid support is quartz.

[0154] Embodiment 29. The method of any one of Embodiments 1 to 28, wherein a first surface of the solid support comprises a layer of polydimethylsiloxane.

[0155] Embodiment 30. The method of Embodiment 29, wherein the polydimethylsiloxane has a ratio of base component to crosslinker component from about 10:1 to about 30:1.

[0156] Embodiment 31. The method of Embodiment 30, wherein the ratio of base component to crosslinker component is about 20:1.

[0157] Embodiment 32. The method of any one of Embodiments 29 to 31, wherein the layer of polydimethylsiloxane has a thickness from about 1 uM to about 120 uM.

[0158] Embodiment 33. The method of Embodiment 32, wherein the layer of polydimethylsiloxane has a thickness from about 20 uM to about 80 uM.

[0159] Embodiment 34. The method of any one of Embodiments 1 to 33, wherein the solid support has a thickness from about 10 µm to about 1 mm.

[0160] Embodiment 35. The method of Embodiment 34, wherein the solid support has athickness from about 100 µm to about 240 µm.

[0161] Embodiment 36. The method of Embodiment 35, wherein the solid support has a thickness of about 170 µm.

[0162] Embodiment 37. The method of any one of Embodiments 1 to 36, wherein the solid support has a length from about 30 mm to about 100 mm and a width from about 40 mm to about 100 mm.

[0163] Embodiment 38. The method of Embodiment 37, wherein the solid support has a length from about 45 mm to about 55 mm and a width from about 60 mm to about 70 mm.

[0164] Embodiment 39. The method of any one of Embodiments 1 to 38, wherein a microfluidic device is adjacent the first surface of the solid support.

[0165] Embodiment 40. The method of Embodiment 39, wherein the microfluidic device is adhered to the polydimethylsiloxane on the first surface of the solid support.

[0166] Embodiment 41. The method of Embodiment 39 or 40, wherein the microfluidic device comprises a plurality of individual spatial areas in a microarray format.

[0167] Embodiment 42. The method of Embodiment 41, wherein the plurality of individual spatial areas have a volume from about 300 picolitres to about 500 picolitres.

[0168] Embodiment 43. The method of any one of Embodiments 39 to 42, wherein the microfluidic device comprises: (a) a first end opposite a second end; (b) a top end and a bottom end, wherein the bottom end is adhered to the solid support; (c) a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the solid support; (d) a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween; (e) a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers; (f) a plurality of control channels configured to open and close the plurality of valves; (g) at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and (h) an outlet disposed at the second end in fluid communication with the network of flow channels.

[0169] Embodiment 44. The method of Embodiment 43, wherein the microfluidic device comprises: (i) a first layer comprising the plurality of control channels, and (ii) a second layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves; wherein the first layer is adhered to the second layer and the second layer isadhered to the solid support.

[0170] Embodiment 45. The method of Embodiment 44, wherein the first layer comprises polydimethylsiloxane and the second layer comprises polydimethylsiloxane.

[0171] Embodiment 46. The method of Embodiment 45, wherein the polydimethylsiloxane in the second layer is crosslinked with the layer of polydimethylsiloxane on the solid support.

[0172] Embodiment 47. The method of any one of Embodiments 43 to 46, wherein the at least one fluidic inlet comprises a plurality of control inlets and a plurality of solution inlets.

[0173] Embodiment 48. The method of Embodiment 47, wherein the plurality of control inlets at the first end of the microfluidic device is in fluid communication with the plurality of control channels.

[0174] Embodiment 49. The method of Embodiment 47 or 48, wherein the plurality of solution inlets is in fluid communication with at least one reagent.

[0175] Embodiment 50. The method of any one of Embodiments 43 to 49, wherein the microfluidic device comprises 1792 chambers, wherein the total number of chambers comprises the plurality of sample chambers and the plurality of reaction chambers.

[0176] Embodiment 51. The method of any one of Embodiments 43 to 50, wherein each sample chamber of the plurality of sample chambers encloses one individual spatial area of the plurality of individual spatial areas on the solid support.

[0177] Embodiment 52. The method of any one of Embodiments 43 to 51, wherein the plurality of valves comprises sandwich valves, button valves, and neck valves.

[0178] Embodiment 53. The method of any one of Embodiments 43 to 52, wherein the outlet is configured to transport waste out of the microfluidic system.

[0179] Embodiment 54. A solid support comprising a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a protease activation sequence, and (iii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to the first surface of the solid support.

[0180] Embodiment 55. The solid support of Embodiment 54, wherein the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain.

[0181] Embodiment 56. The solid support of Embodiment 54 or 55, wherein the protease activation sequence is SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0182] Embodiment 57. The solid support of any one of Embodiments 54 to 56, wherein the protease domain comprises a glutamic protease, an aspartic protease, a cysteine protease, a serine protease, a metalloprotease, or a threonine protease.

[0183] Embodiment 58. The solid support of any one of Embodiments 54 to 57, wherein the protease domain comprises an exopeptidase or an endopeptidase.

[0184] Embodiment 59. The solid support of Embodiment 54 to 58, wherein the protease fusion protein further comprises a detectable N-terminal tag, wherein the detectable N-terminal tag is bound to the protease activation sequence.

[0185] Embodiment 60. The solid support of Embodiment 59, wherein the detectable N- terminal tag is an ALFA-tag glutathione S-transferase, Myc, FLAG-tag, 3X FLAG-tag, HA-tag, polyhistidine-tag, or V5-tag.

[0186] Embodiment 61. The solid support of any one of Embodiments 54 to 60, wherein the detectable substrate is a fluorogenic substrate.

[0187] Embodiment 62. The solid support Embodiment 61, wherein the fluorogenic substrate has an excitation wavelength of about 340 nm or more.

[0188] Embodiment 63. The solid support of any one of Embodiments 54 to 60, wherein the fluorogenic substrate is a FRET substrate.

[0189] Embodiment 64. The solid support of any one of Embodiments 54 to 60, wherein the detectable substrate is a near infrared substrate capable of emitting a near infrared signal.

[0190] Embodiment 65. The solid support of any one of Embodiments 54 to 60, wherein the detectable substrate is capable of emitting a signal detectable by surface-enhanced Raman spectroscopy.

[0191] Embodiment 66. The solid support of any one of Embodiments 54 to 65, wherein the fluorescent protein tag is bound to C-terminus of the protease domain.

[0192] Embodiment 67. The solid support of any one of Embodiments 54 to 65, wherein the fluorescent protein tag is bound to N-terminus of the protease domain

[0193] Embodiment 68. The solid support of any one of Embodiments 54 to 67, wherein the fluorescent protein tag is green fluorescent protein, blue fluorescent protein, cyan fluorescentprotein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein.

[0194] Embodiment 69. The solid support of any one of Embodiments 54 to 67, wherein the fluorescent protein tag is enhanced green fluorescent protein.

[0195] Embodiment 70. The solid support of any one of Embodiments 54 to 69, wherein the fluorescent protein tag is bound to the protease domain via a peptide linker.

[0196] Embodiment 71. The solid support of Embodiment 70, wherein the amino acid linking group is a Gly-Ser amino acid linking group.

[0197] Embodiment 72. The solid support of Embodiment 71, wherein the Gly-Ser amino acid linking group is (Gly4Ser)x, wherein x is an integer from 1 to 10.

[0198] Embodiment 73. The solid support of any one of Embodiments 54 to 72, wherein the fluorescent protein tag is bound to the solid support via an anti-fluorescent protein tag antibody.

[0199] Embodiment 74. The solid support of any one of Embodiments 54 to 72, wherein (i) the fluorescent protein tag is bound to an anti-fluorescent protein tag antibody; (ii) the anti- fluorescent protein tag antibody is bound to a neutralite avid protein; and (iii) the neutralite avid protein is bound to the solid support.

[0200] Embodiment 75. The solid support of Embodiment 73 or 74, wherein the anti- fluorescent protein tag antibody is an anti-fluorescent protein tag variable heavy domain of heavy chain.

[0201] Embodiment 76. The solid support of any one of Embodiments 54 to 75, wherein the solid support is glass.

[0202] Embodiment 77. The solid support of any one of Embodiments 54 to 75, wherein the solid support is quartz.

[0203] Embodiment 78. The solid support of any one of Embodiments 54 to 77, wherein a first surface of the solid support comprises a layer of polydimethylsiloxane.

[0204] Embodiment 79. The solid support of Embodiment 78, wherein the polydimethylsiloxane has a ratio of base component to crosslinker component from about 10:1 to about 30:1.

[0205] Embodiment 80. The solid support of Embodiment 79, wherein the polydimethylsiloxane has a ratio of base component to crosslinker component of about 20:1.

[0206] Embodiment 81. The solid support of any one of Embodiments 78 to 80, wherein thelayer of polydimethylsiloxane has a thickness from about 1 uM to about 120 uM.

[0207] Embodiment 82. The solid support of Embodiment 81, wherein the layer of polydimethylsiloxane has a thickness from about 20 uM to about 80 uM.

[0208] Embodiment 83. The solid support of any one of Embodiments 54 to 82, wherein the solid support has a thickness from about 10 µm to about 1 mm.

[0209] Embodiment 84. The solid support of Embodiment 83, wherein the solid support has a thickness from about 100 µm to about 240 µm.

[0210] Embodiment 85. The solid support of Embodiment 84, wherein the solid support has a thickness of about 170 µm.

[0211] Embodiment 86. The solid support of any one of Embodiments 54 to 85, wherein the solid support has a length from about 30 mm to about 100 mm and a width from about 40 mm to about 100 mm.

[0212] Embodiment 87. The solid support of Embodiment 86, wherein the solid support has a length from about 45 mm to about 55 mm and a width from about 60 mm to about 70 mm.

[0213] Embodiment 88. A microfluidic system comprising a microfluidic device and the solid support of any one of Embodiments 54 to 87.

[0214] Embodiment 89. The microfluidic system of Embodiment 88, wherein the microfluidic device is adjacent the first surface of the solid support.

[0215] Embodiment 90. The microfluidic system of Embodiment 88 or 89, wherein the microfluidic device is adhered to the polydimethylsiloxane on the first surface of the solid support.

[0216] Embodiment 91. The microfluidic system of any one of Embodiments 88 to 90, wherein the microfluidic device comprises a plurality of individual spatial areas in a microarray format.

[0217] Embodiment 92. The microfluidic system of Embodiment 91, wherein the plurality of individual spatial areas have a volume from about 300 picolitres to about 500 picolitres.

[0218] Embodiment 93. The microfluidic system of any one of Embodiments 88 to 92, wherein the microfluidic device comprises: (a) a first end opposite a second end; (b) a top end and a bottom end, wherein the bottom end is adhered to the solid support; (c) a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the solidsupport; (d) a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween; (e) a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers; (f) a plurality of control channels configured to open and close the plurality of valves; (g) at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and (h) an outlet disposed at the second end in fluid communication with the network of flow channels.

[0219] Embodiment 94. The microfluidic system of Embodiment 93, wherein the microfluidic device comprises (i) a first layer comprising the plurality of control channels, and (ii) a second layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves; wherein the first layer is adhered to the second layer and the second layer is adhered to the solid support.

[0220] Embodiment 95. The microfluidic system of Embodiment 94, wherein the first layer comprises polydimethylsiloxane and the second layer comprises polydimethylsiloxane.

[0221] Embodiment 96. The microfluidic system of Embodiment 95, wherein the polydimethylsiloxane in the second layer is crosslinked with the layer of polydimethylsiloxane on the solid support.

[0222] Embodiment 97. The microfluidic system of any one of Embodiments 93 to 96, wherein the at least one fluidic inlet comprises a plurality of control inlets and a plurality of solution inlets.

[0223] Embodiment 98. The microfluidic system of Embodiment 97, wherein the plurality of control inlets at the first end of the microfluidic device is in fluid communication with the plurality of control channels.

[0224] Embodiment 99. The microfluidic system of Embodiment 97 or 98, wherein the plurality of solution inlets is in fluid communication with at least one reagent.

[0225] Embodiment 100. The microfluidic system of any one of Embodiments 93 to 99, comprising 1792 chambers, wherein the total number of chambers comprises the plurality of sample chambers plus the plurality of reaction chambers.

[0226] Embodiment 101. The microfluidic system of any one of Embodiments 93 to 100, wherein each sample chamber of the plurality of sample chambers encloses one individual spatial area of the plurality of individual spatial areas on the solid support.

[0227] Embodiment 102. The microfluidic system of any one of Embodiments 93 to 101, wherein the plurality of valves comprises sandwich valves, button valves, and neck valves.

[0228] Embodiment 103. The microfluidic system of any one of Embodiments 93 to 102, wherein the outlet is configured to transport waste out of the microfluidic system.

[0229] Embodiment 104. The microfluidic system of any one of Embodiments 88 to 103, further comprising an activating protease.

[0230] Embodiment 105. The microfluidic system of Embodiment 104, wherein the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease.

[0231] Embodiments S1 to S49

[0232] Embodiment S1. A glass slide comprising: a glass slide coated with a PDMS polymer on a first surface; a plurality of individual spatial areas in a microarray format on the PDMS polymer on the first surface of the glass slide; and an avidin protein bound to the PDMS polymer and bound to a biotinylated anti-tag protein antibody; wherein each of the plurality of individual spatial areas comprises the avidin protein and a plurality of plasmids encoding an enzyme fusion protein; wherein the enzyme fusion protein comprises an enzyme domain and a protein tag domain, wherein the protein tag domain is capable of binding to the biotinylated anti-tag protein antibody.

[0233] Embodiment S2. The glass slide of Embodiment S1, wherein the PDMS polymer comprises a layer having a thickness of 20 uM to 80 uM.

[0234] Embodiment S3. The glass slide of Embodiment S1 or S2, wherein the PDMS polymer comprises a 20:1 A to B ratio.

[0235] Embodiment S4. The glass slide of any one of Embodiments S1 to S3, wherein each of the plurality of individual spatial areas has a volume between 375 picoliter and 425 picoliter.

[0236] Embodiment S5. The glass slide of any one of Embodiments S1 to S4, wherein the avidin protein comprises a neutralite avidin protein and is non-covalently bound to the biotinylated anti-tag protein antibody through a biotin-avidin interaction.

[0237] Embodiment S6. The glass slide of any one of Embodiments S1 to S5, wherein the biotinylated anti-tag protein antibody comprises an anti-GFP nanobody.

[0238] Embodiment S7. The glass slide of any one of Embodiments S1 to S6, wherein the protein tag domain comprises green fluorescent protein.

[0239] Embodiment S8. The glass slide of any one of Embodiments S1 to S7, wherein the enzyme domain is bound to the protein tag domain by a Ser-Gly linker.

[0240] Embodiments S9. A microfluidic system comprising: the glass slide of any one of Embodiments 1 to 8; and a microfluidic device sealed to the first surface of the glass slide, the microfluidic device comprising a first end opposite a second end, wherein the microfluidic device comprises: a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the glass slide; a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween; a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers; a plurality of control channels configured to open and close the plurality of valves; at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and an outlet disposed at the second end in fluid communication with the network of flow channels.

[0241] Embodiment S10. The microfluidic system of Embodiment S9, wherein the microfluidic device further comprises a first layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves, the first layer is bound to a second layer comprising the plurality of control channels.

[0242] Embodiment S11. The microfluidic system of Embodiment S10, wherein the first layer and the second layer each comprise PDMS.

[0243] Embodiment S12. The microfluidic system of any one of Embodiments S9 to S11, wherein the at least one fluidic inlet comprises a plurality of control inlets and a plurality of solution inlets.

[0244] Embodiment S13. The microfluidic system of Embodiment S12, wherein the plurality of control inlets at the first end of the microfluidic device is in fluid communication with the plurality of control channels.

[0245] Embodiment S14. The microfluidic system of Embodiment S12, wherein the plurality of solution inlets is in fluid communication with at least one reagent.

[0246] Embodiment S15. The microfluidic system of any one of Embodiments S9 to S14, comprising a total number of chambers of 1792, wherein the total number of chambers comprises the plurality of sample chambers plus the plurality of reaction chambers.

[0247] Embodiment S16. The microfluidic system of any one of Embodiments S9 to S15, wherein each sample chamber of the plurality of sample chambers encloses one individualspatial area of the plurality of individual spatial areas on the glass slide.

[0248] Embodiment S17. The microfluidic system of any one of Embodiments S9 to S16, wherein the plurality of valves comprises a combination of sandwich valves, button valves, and neck valves.

[0249] Embodiment S18. The microfluidic system of any one of Embodiments S9 to S17, wherein the outlet is configured to transport waste out of the microfluidic system.

[0250] Embodiment S19. A glass slide comprising: a glass slide having a length between 45 mm and 55 mm, a width between 60 mm and 70 mm, and a depth of less than 0.3 mm; a plurality of individual spatial areas in a microarray format on a first surface of the glass slide; and an avidin protein bound to the first surface of the glass slide and bound to a biotinylated anti-tag protein antibody; wherein each of the plurality of individual spatial areas comprises the avidin protein and a plurality of plasmids encoding an enzyme fusion protein; wherein the enzyme fusion protein comprises an enzyme domain and a protein tag domain, wherein the protein tag domain is capable of binding to the biotinylated anti-tag protein antibody.

[0251] Embodiment S20. The glass slide of Embodiment S19, wherein the glass slide is coated with a PDMS polymer on the first surface.

[0252] Embodiment S21. The glass slide of Embodiment S20, wherein the PDMS polymer comprises a layer having a thickness of 20 uM to 80 uM.

[0253] Embodiment S22. The glass slide of Embodiment S20 or S21, wherein the PDMS polymer comprises a 20:1 A to B ratio.

[0254] Embodiment S23. The glass slide of any one of Embodiments S20 to S22, wherein the avidin protein is bound to the PDMS polymer on the first surface.

[0255] Embodiment S24. The glass slide of any one of Embodiments S19 to S23, wherein the glass slide comprises soda lime glass.

[0256] Embodiment S25. The glass slide of any one of Embodiments S19 to S24, wherein each of the plurality of individual spatial areas has a volume between 375 picoliter and 425 picoliter.

[0257] Embodiment S26. The glass slide of any one of Embodiments S19 to S25, wherein the enzyme domain is a kinase, a protease, or a polymerase.

[0258] Embodiment S27. The glass slide of any one of Embodiments S19 to S26, wherein the avidin protein comprises a neutralite avidin protein and is covalently bound to the biotinylatedanti-tag protein antibody.

[0259] Embodiment S28. The glass slide of any one of Embodiments S19 to S27, wherein the biotinylated anti-tag protein antibody comprises an anti-GFP nanobody.

[0260] Embodiment S29. The glass slide of any one of Embodiments S19 to S28, wherein the protein tag domain comprises green fluorescent protein.

[0261] Embodiment S30. The glass slide of any one of Embodiments S19 to 2S9, wherein the enzyme domain is bound to the protein tag domain by a Ser-Gly linker.

[0262] Embodiment S31. A method of attaching a biotinylated anti-tag protein antibody to a glass slide comprising: contacting a biotinylated anti-tag protein antibody with an immobilized avidin protein wherein the immobilized avidin protein is attached to a PDMS layer on the glass slide.

[0263] Embodiment S32. the method of Embodiment S31, wherein the biotinylated anti-tag protein antibody comprises an anti-GFP nanobody.

[0264] Embodiment S33. The method of Embodiment S31 or S32, wherein the avidin protein comprises a neutralite avidin protein and is covalently bound to the biotinylated anti-tag protein antibody.

[0265] Embodiment S34. A method for detecting enzyme activity in a microfluidic system, the method comprising: (i) contacting a library of enzyme fusion proteins forming part of a microfluidic system with an enzyme binder comprising a fluorescently-labeled moiety; wherein each enzyme fusion protein comprises an enzyme domain and a protein tag domain; wherein the protein tag domain is bound to a biotinylated anti-tag protein antibody and said biotinylated anti- tag protein antibody is bound to a first surface of a glass slide through an avidin protein; (ii) detecting the fluorescently-labeled moiety, thereby detecting enzyme activity in the microfluidic system.

[0266] Embodiment S35. The method of Embodiment S34, further comprising flowing a substrate that when acted upon by the enzyme fusion protein generates a fluorescent product.

[0267] Embodiment S36. The method of Embodiment S34, wherein the library of enzyme fusion proteins comprises an enzyme domain and a protein tag domain.

[0268] Embodiment S37. The method of any one of Embodiments S34 to S36, wherein the library of enzyme fusion proteins is formed by expressing the plurality of plasmid libraries.

[0269] Embodiment S38. The method of any one of Embodiments S34 to S37, wherein theenzyme binder is a nucleic acid, a protein, or a small molecule.

[0270] Embodiment S39. The method of any one of Embodiments S34 to 3S8, wherein the enzyme binder is an enzyme substrate, an enzyme agonist, or an enzyme antagonist.

[0271] Embodiment S40. The method of any one of Embodiments S34 to S39, wherein the fluorescently-labeled moiety is covalently attached to the enzyme binder.

[0272] Embodiment S41 The method of any one of Embodiments S34 to S39, wherein the fluorescently -labeled moiety is non-covalently attached to the enzyme binder.

[0273] Embodiment S42. The method of any one of Embodiments S34 to S41, wherein the fluorescently-labeled moiety comprises green fluorescent protein.

[0274] Embodiment S43. The method of any one of Embodiments S34 to S42, wherein the first surface of Embodiment S The glass slide is coated with a PDMS polymer having a thickness of 20 uM to 80 uM.

[0275] Embodiment S44. The method of any one of Embodiments S34 to S43, wherein the avidin protein comprises a neutralite avidin protein and is covalently bound to the biotinylated anti-tag protein antibody.

[0276] Embodiment S45. The method of any one of Embodiments S34 to S44, wherein the biotinylated anti-tag protein antibody comprises an anti-GFP nanobody.

[0277] Embodiment S46. The method of any one of Embodiments S34 to S45, wherein the enzyme binder comprises a Ser-Gly linker.

[0278] Embodiment S47. The method of any one of Embodiments S34 to S46, wherein the microfluidic system is formed by aligning a microfluidic device comprising a plurality of chambers with the glass slide, thereby forming a microarray wherein each of the plurality of chambers contains one of the plurality of plasmid libraries.

[0279] Embodiment S48. The method of Embodiment S47, wherein the plurality of chambers comprises a plurality of sample chambers and a plurality of reaction chambers.

[0280] Embodiment S49. The method of any one of Embodiments S47 or S48, wherein the microfluidic device comprises PDMS. EXAMPLES

[0281] Example 1

[0282] Using the techniques described herein, a reliable and improved microfluidic system 1for high-throughput reactions was created. As depicted in FIG.1, the microfluidic system 1 is a microfluidic device 2 bonded to a solid support, such as a glass slide 4.

[0283] In preparing the microfluidic system, other common devices employ the use of epoxy silane coating on the solid support (e.g., glass slide) as an initial layer before arraying the sample. Epoxy silane enables the bonding of PDMS microfluidic devices to bond to the solid support (e.g., glass slide). In a typical protocol, once the microfluidic device is aligned on the epoxy silane slide, the system is baked at 95 °C for 12 hours to cure the bond between the glass and the microfluidic device. As previously described, these cured epoxy silane bonds frequently fail resulting in total loss of the system. Further, the baking process degrades the sample deposited on the glass causing decreased protein expression. The epoxy silane coating was eliminated and instead the solid support (e.g., glass slide) was coated with PDMS.

[0284] To prepare the PDMS, the crosslinker (Part B) and the base (Part A) were weighed out, added together, and mixed for 3 minutes at 2000 rpm in a mixer (e.g., THINKY® AR-100), and then defoamed for 3 minutes at 2200 rpm.

[0285] With reference to FIG.2, to prepare the solid support (e.g., glass slide) 4, the bare solid support (e.g., glass slide) was placed on a pedestal of a spin coater in a laminar flow hood with a quarter sized amount of the prepared PDMS with a 20:1 A (base) to B (crosslinker) ratio deposited on the center of the solid support (e.g., glass slide). The lid of the spin coater was closed and a vacuum was applied to hold the slide to the pedestal. The slide and PDMS were spun for 500 rpm for 10 seconds and 1820 rpm for 1 minute resulting in a PDMS layer about 100 uM thick on the solid support 4 (e.g., glass slide). The solid support 4 was then baked at 80 °C for 40 minutes.

[0286] After coating, the designed plasmid library sample 6 was arrayed on the PDMS layer 5 of the solid support 4 such that a plurality of individual spatial areas 8 was created at known locations, see FIG.2 and Step A of FIG.6. A SCIENION® S3 SciFlexarrayer was used to array the designed plasmid library sample 6. The individual spatial areas 8 were arranged in a microarray 9 across the solid support 4 before assembling into the microfluidic system. The individual spatial areas each had a volume of about 400 picoliters.

[0287] Device Fabrication

[0288] A two-layer PDMS microfluidic device with integrated pneumatic valves was aligned on the solid support (e.g., glass slide). To prepare the microfluidic device, a thick control layer and a thin flow layer were aligned with one another and baked. For the control layer, the PDMSwas prepared by weighing out the crosslinker (Part B) and the base (Part A) in a 5:1 A to B ratio. The two components are combined and added to a THINKY® mixer to mix for 3 minutes at 2000 rpm and defoamed for 3 minutes at 2200 rpm. About 55 grams of the mixed PDMS is deposited on a silicon wafer flow layer molding master (previously etched with the mold of the control layer using standard photolithography procedures) and placed in a vacuum chamber for degassing. The vacuum chamber is closed and a pump is applied for 45 minutes.

[0289] For the flow layer, the PDMS was prepared by weighing out the crosslinker (Part B) and the base (Part A) in a 20:1 A to B ratio. The two components were combined and added to a THINKY® mixer to mix for 3 minutes at 2000 rpm and defoamed for 3 minutes at 2200 rpm. A 1.5” diameter circle of the mixed PDMS was placed on a silicon wafer flow layer molding master (previously etched with the mold of the flow layer using standard photolithography procedures) on a spin coater. The spin coater was programed to spin at 500 rpm for 10 seconds and 1750 rpm for 75 seconds. Once the spin coater was done, the wafer rested for 5 minutes.

[0290] The control layer was baked at 80 °C for 50 minutes in a convection oven and the flow layer was baked at 80 °C for 40 minutes in a convection oven. The control layer was peeled off the wafer and holes were punched into the PDMS with a drill press. The control layer and the flow layer were aligned using a microscope and both layers were baked together at 80 °C for 50 minutes. Holes were then punched into the flow layer using similar techniques.

[0291] With reference to FIG.3 and Step B of FIG.6, the microfluidic device was aligned over the solid support 4 such that each of the individual spatial areas 8 was sealed within an individual chamber within the microfluidic device 2. As further depicted in FIG.4, the microfluidic device 2 contained a plurality of sample chambers 15 in fluid communication with a plurality of reaction chambers 17. Once the microfluidic device 2 was positioned, the microfluidic system 1 was baked on a hot plate at 80 °C for 6-8 hours, see Step C of FIG.6. Compared to systems with epoxy silane that required baking on a hot plate at 95 °C for 12 hours, the PDMS and solid support (e.g., glass slide) bond failed less frequently, and the microfluidic systems exhibited improved protein expression.

[0292] The solid support (e.g., glass slide) had a thickness of about 170 µm. In systems with a 170 µm thick solid support (e.g., glass slide), the signal to noise ratio as background fluorescence of the glass decreased, thereby improving the results of the assay.

[0293] The microfluidic device can take on many configurations, but generally the microfluidic device, such as depicted in FIG.5, is made of two PDMS layers. The first layer hasa plurality of sample chambers, reaction chambers, and valves. Sample chambers enclose the individual spatial area of sample, and reaction chambers are in fluid communication with the sample chambers. The second layer of the device contains a plurality of control channels in communication with the valves. The control channels open and close the valves when there is a change in pressure within the device. Inlets are positioned on one end of the device and one or more outlets in communication with the flow channels are positioned at an opposing, second end of the device. Reagents enter the device and access the chambers through the inlets and exit the device through the at least one outlet. The inlets, as depicted in FIG.5, are a plurality of control inlets in communication with the plurality of control channels and a plurality of solution inlets. Solution inlets transfer reagents, buffers, and water into the fluidic device. In embodiments, the total number of chambers is 1792, however fluidic devices with more or less chambers can be made.

[0294] High-Throughput Microfluidic Enzyme Kinetics

[0295] In typical protocols, once the solid support (e.g., glass slide) and device are assembled, a region of each chamber is functionalized with anti-fluorescent protein tag antibody bound to a neutralite avidin protein, where the neutralite avid protein is adjacent the first side of the glass side. The anti-fluorescent protein tag antibody bound to a neutralite avidin non-specifically sticks to the PDMS of the microfluidic device. The fluorescent protein tag expressed by the plasmid is pulled down when exposed to the anti-fluorescent protein tag antibody. Due to the PDMS coated glass, the neutralite avidin protein nonspecifically sticks to the PDMS surface of the solid support without further functionalization. This reduces preparation time, in some instances over 1 hour, and decreases cost of the functionalization step, in some cases by about 30%.

[0296] As depicted in FIG.7, an anti-fluorescent protein tag antibody is attached to the solid support (e.g., glass slide) by a neutralite avidin protein. The neutralite avidin protein is both bound to the PDMS and non-covalently bound to the anti-fluorescent protein tag antibody. The anti-fluorescent protein tag antibody provide for attachment of the protease fusion protein via the fluorescent protein tag in the protease fusion protein. Target protease fusion protein variants are expressed using cell-free expression methods, such as cell-free lysates or in vitro protein synthesis systems. PUREXPRESS® in vitro protein synthesis kits are purchased from New England Biolabs.

[0297] To apply the anti-fluorescent protein tag antibody bound to the neutralite avidin protein, control line pressure was increased to 40-45 psi such that the button valves and the neckvalves were closed within the device. Neutralite avidin protein was then inserted into the inlet and flowed through at 3-4 psi flow line pressure for 30 minutes. Phosphate-buffered saline (PBS) was then applied at the inlets and flowed through the device for 30 minutes. The button valves were opened and neutravidin flowed through the device for 30 minutes. PBS was then applied at the inlets and flowed through the device, with the button valves open, for 15 minutes. The anti-fluorescent protein tag antibody in PBS flowed through the device for a few minutes followed by another PBS wash for 15 minutes with the button valves closed. The device was then washed with an enzyme-specific reaction buffer for 15 minutes and the device was loaded onto a microscope for imaging.

[0298] Example 2

[0299] Mechanistic Enzyme Kinetics (HT-MEK) for the kinetic characterization of protease function (HT-MEKpro) enables the measurement of quantitative biochemical constants describing protease activity (e.g., kcatand KM) for libraries of up to 102–103protease variants or more (FIG.7). See Markin et al, Science 373:6553 (2021). The first focus was on Mpro, a 3C- like cysteine protease that cleaves SARS-CoV-2 polyproteins to generate 12 non-structural proteins Nsp4-Nsp16. To adapt Mprofor on-chip (i.e., microfluidic) studies the inventors identified successful expression conditions and constructs, compatible substrates for on-chip assays, and showed accurate measurements for WT Mproactivity and inhibition on-chip. Thisassay was applied to an initial library of 400 Mpro variants, identifying mutations leading tohyperactivity, sites sensitive to mutations, and residue changes conferring resistance. This was extended to on-chip studies to fourteen orthologous 3C-like proteases from other viruses, showing that differences in their catalytic activities and inhibitions with nirmatrelvir and ensitrelvir were identified.

[0300] Results

[0301] HT-MEK enabled the expression, purification, and assay of thousands of enzyme variants per device and was used to measure the multiple kinetic and thermodynamic parameters (e.g., kcat / KM for multiple substrates and Ki for product inhibition) of 1036 mutants of the model enzyme PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium). HT-MEK utilized a two-layer microfluidic device composed of polydimethylsiloxane (PDMS). Each device contained 1792 chambers that were separated from one another by a built-in pneumatic valve (sandwich). Each chamber was divided by a second valve (neck); one half was used for enzyme expression (containing a spot of DNA encoding for a known enzyme variant) and the other half for enzyme assay. A third valve (button) served two functions: (1) it reversiblyprotected a circular patch in the reaction chamber for surface patterning of an anti-eGFP nanobody, which pulled down expressed enzyme variants by their eGFP tag and allowed for their purification via continuous flow with compatible buffers, and (2) it enabled the reversible shielding of the expressed enzyme, such that each substrate-filled chamber was exposed to enzyme in parallel and turnover measured via fluorescent microscopy. HT-MEK was adapted for the study of proteases (HT-MEKpro), first focusing on Mprofrom SARS-CoV-2 because of ongoing drug discovery efforts and concerns of resistance and then applying these methods to fourteen orthologous Mprofrom related viruses (FIG.7).

[0302] Construct optimization

[0303] Proteases are often expressed as zymogens (i.e., an inactive precursor enzyme) to prevent aberrant proteolysis and enable precise protease regulation and signaling. Conversion to the active recombinant enzyme typically involves removal of an activation segment, often an N- terminal extension of the protease, by either autolytic cleavage or cleavage by an orthogonalprotease (tobacco etch virus (TEV) protease, enterokinase (EK), 3C protease, etc.). SARS-CoV-2 Mprois expressed as a part of the viral polyprotein, and will self-cleave at the N- and C-termini releasing active Mproto then cleave the remaining NSP sites within the viral polyprotein. This auto-activation strategy has been previously utilized in heterologous expression.

[0304] SARS-CoV-2 Mpro was engineered into an eGFP-fusion construct (FIG.8A). The eGFP-tagged was linked to the C-terminus of the enzyme through a Gly-Ser (G4S)5 linker as the native N-termini is crucial in dimer formation and activity. Since the C-terminus of Mpro is a native substrate at Q306, all constructs contained a Q306A mutation to prevent any self-cleavage from the eGFP tag. Engineering efforts were focused on the N-terminus of the enzyme, as the N- terminal tag and activation sequence can have dramatic effects on expression and activation of the fusion protein. A series of N-terminal tags, including glutathione S-transferase (GST), Myc, FLAG, and 3X FLAG, and activation sequences (Mpro(auto-activation), enterokinase (EK), Tobacco Etch Virus (TEV)) were tested for expression and activation of the enzyme on device (FIG.8A).^Other useful N-terminal tags included HA, His, and V5.

[0305] Initially efforts were focused on the activation strategy to see which strategy yielded complete activation of the enzyme. Three strategies: auto-activation, EK, and TEV were explored. Each construct was GST-tagged and contained the corresponding activation sequence for each strategy to release the native enzyme. Additionally, constructs mutating P1 cleavage residue in the activation sequence and / or the catalytic cysteine of Mpro-eGFP Cys145 to alanine were cloned to see how tightly regulated activation is by the activating enzyme. Althoughrequiring less steps, activation by an auto-activation approach would be dependent on the yield of the enzyme and the activity of the enzyme variant. When expressed in PUREXPRESS®, auto-activation of WT enzyme can be observed; however, catalytically inactive enzyme (C145A) was unable to activate. If the auto-activation approach was expanded to variants with unknown function, this would lead to concerns of if the enzyme is catalytically inactive or unable to activate itself (FIG.8B).^

[0306] Supplementing in an unrelated activating protease should allow for complete activation regardless of the yield and activity of the expressed enzyme. Initial attempts with enterokinase activation did completely remove the GST tag, however, due to an unknown internal cut site, treatment with enterokinase led to a secondary byproduct. By using TEV, we demonstrated complete activation of Mpro-eGFP with both WT and C145A. When the P1 glutamine in the cut site was mutated to alanine, activation was diminished (FIG.8B).^

[0307] Activity of the expressed enzyme was checked using a commercially Rh110- conjugated substrate SEQ ID NO:8 (where Rh110 is conjugated to SEQ ID NO:8). Activity of the enzyme was visualized by the increase in fluorescence (485 / 535) by plate reader through the release of Rh110 via proteolysis after glutamine. Expressed WT Mpro-eGFP activated by TEV demonstrated activity on the fluorogenic substrate (FIG.2C). When treated with nirmatrelvir, activity was inhibited. Additionally, when Mpro-eGFP was expressed without TEV, the protease- fusion remained inactive and did not cleave the substrate. When the catalytically inactive mutant (C145A) was expressed and activated by TEV, no activity was measured. Neither TEV nor PUREXPRESS® demonstrated any activity against the fluorogenic substrate.^Additionally, we demonstrated that excess PUREXPRESS® is non-inhibitory to the enzyme (FIG.8C).

[0308] Initial attempts at expressing GST-tagged constructs of Mpro-eGFP on device resulted in unquantifiable expression. The N-terminal tag proved important for improving expression levels on-device. FLAG, Triple-FLAG, and Myc tags were tested resulting in the FLAG-tagged constructs yielding 1-5 nM enzyme on device. Triple-FLAG-tagged constructs maxed out expression around 1 nM and Myc-tagged constructs were unquantifiable, similar to GST. Unlike off-device expression, Adding TEV to the expression mixture on device, greatly reduced protein yields which led to a two-step process of expression then activation (FIG.8D).

[0309] The activation of the enzyme can be checked using a DYLIGHT®550-labeled anti- FLAG antibody at the end of the experiment. Initial attempts to add antibody between activation and activity assays, inhibited all activity (data not shown). The activation of the enzymes can be quantified using a ratio of Ab fluorescence to eGFP signal. Using this strategy, WT FLAG-Mpro-eGFP can auto-activate itself at the TEV site regardless of if TEV is added or not. The differences between the auto-activation of the GST versus FLAG constructs may be due to the overall yield and / or steric hindrance of the activation site. As expected, both constructs with glutamine in the TEV site mutated to an alanine did not completely activate as evidenced by antibody binding to the button post TEV activation. This may be due to promiscuous activity from WT Mpro on the TEV cleavage site. After a 2 hr activation with TEV, the signal ratio of catalytically inactive (C145A) of Mpro-eGFP demonstrated a large drop in signal indicating complete removal of the N-terminal tag (FIG.8E).^

[0310] Rational design of a compatible fluorogenic substrate for Mproon HT-MEK

[0311] Fluorescence signal accumulation can be visualized and quantified in chambers containing wild-type enzyme. Similarly to off-device kinetics, on-chip activity can be quantified using the same Rh110 substrate, (SEQ ID NO:8)-Rh110 (FIGS.8F-8G). However, initial attempts to apply the Rh110-labeled substrate that was used for off-device activity assays demonstrated limitations in quantifying Michaelis-Menten kinetics due the solubility of the substrate. This motivated us to pursue a new substrate for on-device activity measurement.

[0312] There is a wealth of fluorophores that can be applied to the design and synthesis of protease fluorogenic substrates. Typically, these protease fluorescent substrates work in either one of two ways. First, the cleavage of an amide bond between a peptide and an inactivated fluorophore, and upon cleavage, the fluorophore is able to fluoresce. Second, an internally quenched peptide substrate with a fluorophore and quencher conjugated off of the N and C- termini, and upon cleavage, the release of the fluorophore-peptide fragment allowing for fluorescence.

[0313] For the former strategy, fluorophores including AMC, ACC, and Rh110 can be applied. Initial attempts with AMC^demonstrated the fluorophore’s permeability into the PDMS. Through flow on experiments, AMCA, ACC, and Rh110 appeared to be polar enough to remain within the chambers. Typically, these substrates which release the active fluorophore exhibit weaker catalytic activity due to unnatural scissile bond and weaker KM values thus limiting the dynamic range of activity that can be quantified, especially on the less catalytically efficient enzymes. Additionally, these types of substrates are challenging to synthesize.

[0314] The inventors pursued the latter strategy of internally quenched peptides for on-device activity measurements of Mpro. There are many established internally quenched peptide substrates for SARS-CoV-2 Mprothat are typically designed based on the P4-P4’ of NSP4-5 andNSP7-8 cleavage sites of SARS-CoV-2; however, these also led to complications of solubility of substrates due to the notorious hydrophobic nature of NSP cleavage sites. We developed a device compatible substrate that overcame the solubility issues by installing the P6-P6’ of the NSP4-5 cleavage site with a 5-FAM conjugated lysine and a DNP-conjugated lysine on the N- and C-termini respectively. The additional asparagine and lysine at P6-P5 and additional glutamic acid at P5’ enhanced the solubility, thereby preventing apparent aggregation.

[0315] Through timelapse microscopy, the turnover of the fluorescent substrate per chamber was quantified to get individual rates per normalized to enzyme concentration. When 30 µM substrate was flown onto the device for WT Mproan increase in fluorescent product was quantified. For the C145A variant, which is catalytically inactive, no turnover was observed. When 30 µM substrate was flowed on with 20 µM ensitrelvir (an established Mproinhibitor) activity was blocked. Additionally, both WT and C145A were fully activated using TEV since the ⍺-FLAG antibody did not bind. In a C145A construct with the TEV cleavage site’s glutamine mutated to an alanine, which was visualized by the presence of the tag as a positive control.

[0316] HT-MEK can reproduce kinetic parameters quantified through traditional assays

[0317] To benchmark how quantitative or qualitative protease kinetic measurements are on HT-MEK, four variants of SARS-CoV-2 Mprowere used to benchmark parameters to off-device values. The four variants selected were wild-type (WT), P132H (a high frequency mutation in the population), L050F (a pre-established hyperactive mutation), and E166V (a catalytically slow mutation demonstrating increased resistance to nirmatrelvir). Michaelis-Menten kinetic and inhibition assays were done for each variant on and off device.

[0318] On-device Michaelis-Menten assays were run through a series of 10 assays of increasing substrate concentrations from 0.4 µM to 200 µM. The serial dilutions were done in DMSO followed by a subsequent dilution into buffer to standardize concentration of DMSO across all samples as small amounts of DMSO can have substantial effects on Mproactivity. Each assay was reduced to 15 minutes: 5 minute flow-on of next substrate concentration with buttons closed followed by a 10 min activity assay with buttons on. Using our codebase, images can be background subtracted, initial rates can be quantified, and the rates can be fit for Michaelis-Menten parameters (kcatand KM). Each chamber will be individually fit for Michaelis- Menten parameters followed by averaging across all chambers with identical variants.

[0319] HT-MEK can reproduce inhibition trends from inhibition assays.

[0320] On-device inhibition assays were run similarly to on-device Michaelis-Menten assays with a series of 12 assays of increasing inhibitor concentration. The inhibitor concentration range can be adjusted based on expected IC50 for WT SARS-CoV-2 Mpro, but as all inhibitors in this study have demonstrated Ki in the low nM range an inhibitor dilution series from 20 pm to 90 µM was used. Each assay was 15 minutes: 5 minute flow-on of next inhibitor concentration with buttons closed followed by a 10 min activity assay with buttons open. A secondary codebase was developed to process inhibition values using initial rates to calculate percent inhibition and fit those values to a dose response curve. The IC50 values were converted to Ki using the Cheng-Prusoff Correction as the KMwas previously quantified on device. Two pre- established inhibitors were used to benchmark inhibition values: nirmatrelvir (a covalent active site inhibitor which is the active component of Paxlovid) and ensitrelvir (a non-covalent active site inhibitor).

[0321] Mpro-eGFP fusion scaffold is applicable to other Mpro from other coronaviruses

[0322] To demonstrate translatability of the scaffold not only to mutations, but to related viral proteases, 14 other viral proteases from other coronaviruses were cloned into the eGFP-fusion scaffold. The proteases were selected from multiple genera of coronaviruses (alpha, beta, gamma, delta, and epsilon) and multiple species (human, porcine, avian, mice, bat, and feline). As all the selected proteases have a Ser or Ala as the first amino acid of the native sequence, the TEV activation strategy did not need to be adjusted.

[0323] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

Claims

CLAIMS What is claimed is:

1. A solid support comprising a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises: (a) a protease domain, (b) a protease activation sequence, and (c) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to the first surface of the solid support.

2. The solid support of claim 1, wherein the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain.

3. The solid support of claim 1, wherein a first surface of the solid support comprises a layer of polydimethylsiloxane.

4. The solid support of claim 3, wherein the polydimethylsiloxane has a ratio of base component to crosslinker component from about 10:1 to about 30:

1.

5. A microfluidic system comprising a microfluidic device and the solid support of claim 1.

6. The microfluidic system of claim 5, wherein the microfluidic device is adjacent the first surface of the solid support.

7. The microfluidic system of claim 5, wherein the microfluidic device is adhered to the polydimethylsiloxane on the first surface of the solid support.

8. The microfluidic system of claim 5, wherein the microfluidic device comprises: (a) a first end opposite a second end; (b) a top end and a bottom end, wherein the bottom end is adhered to the solid support; (c) a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the solid support; (d) a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween;(e) a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers; (f) a plurality of control channels configured to open and close the plurality of valves; (g) at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and (h) an outlet disposed at the second end in fluid communication with the network of flow channels.

9. The microfluidic system of claim 5, wherein the microfluidic device comprises (a) a first layer comprising the plurality of control channels, and (b) a second layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves; wherein the first layer is adhered to the second layer and the second layer is adhered to the solid support.

10. The microfluidic system of claim 9, wherein the first layer comprises polydimethylsiloxane and the second layer comprises polydimethylsiloxane.

11. The microfluidic system of claim 10, wherein the polydimethylsiloxane in the second layer is crosslinked with the layer of polydimethylsiloxane on the solid support.

12. A method for assessing protease activity in a microfluidic system, the method comprising: (a) reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises: (i) a protease domain, (ii) a protease activation sequence, wherein the protease activation sequence is bound to the protease domain, and (ii) a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a first surface of a solid support; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein;(b) reacting the activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; and (c) detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

13. The method of claim 12, wherein the activating protease is a tobacco etch virus protease, an enterokinase, or a SARS-CoV-23CL protease.

14. The method of claim 12, wherein the amino acid sequence of the protease activation sequence is orthogonal to the amino acid sequence of the protease domain.

15. The method of claim 12, wherein the protease fusion protein further comprises a detectable N-terminal tag, wherein the detectable N-terminal tag is bound to the protease activation sequence.

16. The method of claim 12, further comprising, prior to step (a), expressing a plasmid to produce the protease fusion protein.

17. A method for assessing protease activity in a microfluidic system, the method comprising: (a) reacting an activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; wherein the activated protease fusion protein comprises a protease domain bound to a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to a solid support; and (b) detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

18. The method of claim 12, wherein a first surface of the solid support comprises a layer of polydimethylsiloxane.

19. The method of claim 18, wherein the polydimethylsiloxane has a ratio of base component to crosslinker component from about 10:1 to about 30:

1.

20. The method of claim 12, wherein a microfluidic device is adjacent the first surface of the solid support.

21. The method of claim 20, wherein the microfluidic device is adhered to thepolydimethylsiloxane on the first surface of the solid support.

22. The method of claim 20, wherein the microfluidic device comprises: (a) a first end opposite a second end; (b) a top end and a bottom end, wherein the bottom end is adhered to the solid support; (c) a plurality of sample chambers configured to enclose the plurality of individual spatial areas on the solid support; (d) a plurality of reaction chambers in fluid communication with the plurality of sample chambers by a network of flow channels therebetween; (e) a plurality of valves configured to control fluid movement between the plurality of sample chambers and the plurality of reaction chambers; (f) a plurality of control channels configured to open and close the plurality of valves; (g) at least one fluidic inlet disposed at the first end in fluid communication with the network of flow channels; and (h) an outlet disposed at the second end in fluid communication with the network of flow channels.

23. The method of claim 20, wherein the microfluidic device comprises (a) a first layer comprising the plurality of control channels, and (b) a second layer comprising the plurality of sample chambers, the plurality of reaction chambers, and the plurality of valves; wherein the first layer is adhered to the second layer and the second layer is adhered to the solid support.

24. The method of claim 23, wherein the first layer comprises polydimethylsiloxane and the second layer comprises polydimethylsiloxane.

25. The method of claim 24, wherein the polydimethylsiloxane in the second layer is crosslinked with the layer of polydimethylsiloxane on the solid support.

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