Non-ionic surfactants and methods of using the same

Abstract

Disclosed herein are compounds that can be used as surfactants for various applications. The disclosed compounds are biodegradable and do not form toxic by-products when degraded and can be used as replacements for current surfactants, such as octylphenol ethoxylates (OPEs) and nonylphenol ethoxylates (NPEs) that have toxic effects when degraded. Surfactant-based compositions, methods, kits, and systems can be used in cell permeabilization, analytical / diagnostic assays, polypeptide stabilization, viral inactivation, and bioprocess workflows. Permeabilization buffers comprising the compounds, permeabilize diverse cell types while preserving intracellular targets and are compatible with a variety of downstream detection and analysis methods. Compositions include loading, blocking, running, mountant buffers, polypeptide stabilization and viral inactivation solutions. The surfactants are used in lateral flow devices, bead-based assays, and lipid nanoparticle workflows. The disclosure further provides methods and systems in which data generated from surfactant-containing workflows are analyzed by machine-learning processes to determine attributes performance and recommend protocol parameters.

Description

NON-IONIC SURFACTANTS AND METHODS OF USING THE SAME CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U. S. C. §119(e) to U. S. Provisional Application No. 63 / 739,959 filed on December 30, 2024, the contents of which are hereby expressly incorporated herein by reference in their entirety as though fully set forth herein.FIELD

[0002] The present disclosure is directed to new biodegradable surfactants and methods of making and using the same.BACKGROUND

[0003] Currently available non-ionic surfactants, such as Triton™ X-100 and Tergitol™ NP-40, are part of a group of chemicals known as octylphenol ethoxylates (OPEs) and nonylphenol ethoxylates (NPEs) that, when broken down in the environment, have toxic effects on aquatic organisms at extremely low concentrations. OPEs and NPEs are toxic xenobiotic compounds classified as endocrine disrupters capable of interfering with the hormonal system of numerous organisms. The use of OPEs and NPEs has already been banned for most uses in the EEA, UK, and Switzerland requiring capture, diversion, and incineration of all waste with limited exemptions or authorization. Many other countries are evaluating similar actions where exemptions or authorizations are less clear than in the EU. There exists a need in the art for new surfactants that are biodegradable and do not exhibit the toxic effects associated with currently available non-ionic surfactants.SUMMARY

[0004] Disclosed herein is a compound having a structure according to Formula I,R4FY’Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

[0005] Also disclosed is a method, comprising exposing a sample to a compound according to the present disclosure. Also disclosed is a method of lysing a cell, comprising contacting the cell with an amount of a compound according to the present disclosure sufficient to facilitate cell lysis.

[0006] Also disclosed is a composition, comprising a compound according to the present disclosure. Also disclosed is a kit, comprising a compound according to the present disclosure; and a container.

[0007] The present disclosure relates to amphiphilic surfactant compounds and their use in a variety of biological compositions, methods, devices, and kits. In particular, the disclosure provides surfactants having a general structure according to Formula I and related structures (including Formulas I A— IK and defined compounds 1-54) and the use of such surfactants in cell permeabilization buffers, electrophoresis and immunoassay buffers, blocking buffers, mountants,lateral flow assay components, polypeptide stabilization formulations, viral inactivation compositions, and lipid nanoparticle (LNP) analysis reagents. The surfactants are formulated in polar protic solvents, optionally together with inorganic salts, buffering agents, and chaotropic agents, and are used at concentrations effective to achieve desired biological functions such as permeabilization, stabilization, blocking, improved flow, or viral inactivation.

[0008] The surfactant compounds are according to Formula I;wherein: R1is a hydrophilic group; R2is a lipophilic group; X is selected from oxygen, sulfur, or N(R5); R3is selected from heteroaliphatic, aliphatic, or aryl; each R4independentlyis aliphatic; Y is selected from oxygen, sulfur, or N(R5); the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5; n is an integer selected from 0 or 1; m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1; p is an integer selected from 0 to 2; and each R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic. In some embodiments, the surfactants are nonionic.

[0009]

[0010] In one aspect, the disclosure provides cell permeabilization buffers comprising (a) a surfactant according to Formula I, as described herein; and (b) a polar protic solvent.

[0011] In one aspect, the disclosure provides cell permeabilization buffers comprising (a) a surfactant selected from Formulas IA— IF, IG— IK, as described herein; and (b) a polar protic solvent.

[0012] In one aspect, the disclosure provides cell permeabilization buffers comprising (a) a surfactant selected from compounds 1-54 as described herein; and (b) a polar protic solvent. In cell permeabilization buffers of the disclosure as described in the various aspects, a surfactant as described above is typically present at a concentration between about 0.005% and 5% (v / v), in an amount sufficient to permeabilize cells.

[0013] Cell permeabilization buffers of the disclosure can optionally further comprises one or more of the following: inorganic salts, buffering agents, blocking agents, fixative quenchers, enzymes, and / or chaotropic agents and various combinations thereof. Exemplary fixative quenchers include glycine, ammonium chloride, Tris, ethanolamine, lysine, sodium borohydride, serum, and bovine serum albumin (BSA). Exemplary blocking agents include albumin, casein, gelatin, and BSA. In some embodiments, proteases or cell wall-lytic enzymes, such as trypsin, proteinase K, pepsin, pronase, papain, lysozyme, zymolyase, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, glucanase, collagenase, or dispase, are included in the permeabilization buffer.

[0014] The present disclosure also provides methods for permeabilizing cells comprising contacting cells with a permeabilization buffer of the disclosure comprising an amount of the surfactant effective to facilitate permeabilization of the cell. The methods are applicable to a wide range of cell types including eukaryotic cells, plant cells, fungal cells, and bacterial cells, and to specific cell populations such as blood cells, immune cells, cultured cells, and primary cells. The permeabilized cells can be used in a variety of methods for detecting one or more analytes found in the cells, where a biological sample comprising a cell, is contacted with the permeabilization buffer for a time sufficient to permeabilize the cells, optionally in conjunction with a fixation buffer. The permeabilized sample is then incubated with one or more analyte binding agents (e.g., antibodies, antibody fragments, aptamers, or oligonucleotides) that can enter the cell and bind intracellular or extracellular analytes. Detection can be performed directly, using analyte binding agents that carry detectable labels, or indirectly, using secondary analyte binding agents or reagents that generate a detectable signal upon binding. The analytes may include proteins, nucleic acids, carbohydrates, lipids, metabolites, or combinations thereof.

[0015] In some embodiments, methods of the disclosure can be carried out in a singleplex format. In some embodiments, methods of the disclosure can be carried out in a multiplex format, employing multiple analyte binding agents that specifically bind different analytes, and can involve repeated rounds of binding, washing, and detection. In certain embodiments, the analysis further comprises nucleic acid polymerization and / or amplification using a polymerase and / or transcriptase.

[0016] Kits are provided that comprise a cell permeabilization buffer of the disclosure and one or more additional reagents selected from fixation buffers, mountants, wash buffers, analyte binding agents, gels, matrices, membranes, and combinations thereof. The kits enable standardizedworkflows for cell permeabilization, intracellular staining, and downstream analysis in research or diagnostic settings.

[0017] In another aspect, the disclosure provides compositions for electrophoresis and immunoassays. In one embodiment, there is provided a loading buffer for gel electrophoresis of proteins or nucleic acids comprising a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54), a tracking dye, and optionally glycerol and / or a reducing agent. In a related embodiment, the surfactant is used in the manufacture or formulation of tracking dyes for gel electrophoresis.

[0018] The disclosure further provides blocking buffers comprising a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54), at least one polar protic solvent, optional inorganic salt, optional buffering agent, and optional chaotropic agent; a tracking dye; a blocking agent (e.g., non-fat dry milk, albumin, BSA, casein, gelatin); and a buffering agent. Kits containing such blocking buffers, optionally together with wash buffers, gels, transfer membranes, lateral flow strips, and analyte binding agents, are configured such that the surfactant concentration is effective to inhibit non-specific interactions between polypeptides while maintaining specific binding between analytes and analyte binding agents. Methods are disclosed for reducing nonspecific binding of antibodies or antibody fragments to membranes or matrices (such as nitrocellulose membranes, PVDF membranes, or affinity column matrices) in assays including Western blots, ELISA assays, dot blots, Northern blots, Southern blots, and lateral flow assays, by contacting the membrane or matrix with the blocking buffer.

[0019] The disclosure also provides mountant compositions comprising a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54); at least one polar protic solvent; optional inorganic salts, buffering agents, and chaotropic agents; and a base medium. The base medium can include one or more of an antifade or fluorophore stabilizer, refractive index modifier, preservative, hardening agent, wetting agent, or clearing agent. The surfactant-containing mountants are suitable for imaging applications, and the surfactant is used in the manufacture of such mountants. Compositions are provided that include beads or microcarriers conjugated to analyte binding agents in the presence of surfactants according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54). Kits are disclosed that include capture antibody reagents (beads or microcarriers conjugated to firstanalyte binding agents specific for a first epitope on a target analyte), detection antibody reagents (second analyte binding agents labeled with detectable labels and specific for a second epitope on the same analyte), and optional lysis and wash buffers. One or more of the capture reagent, detection reagent, lysis buffer, or wash buffer includes a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54). In some embodiments, capture beads are internally labeled with dyes, and a plurality of capture / detection reagent pairs is provided to detect multiple target analytes (e.g., 2 to 100 analytes). Detectable labels can include radiolabels, organic fluorophores, fluorescent proteins, quantum dots, chromophores, enzymatic labels, chemiluminescent labels, bioluminescent labels, metal-based labels, oligonucleotide-based labels, affinity tags, haptens, detectable substrates, or colorimetric protein or peptide detection dyes.

[0020] In additional embodiments, the disclosure provides kits for nucleic acid-based analyte detection that exploit oligonucleotide probes. Such kits comprise analyte capture reagents including analyte binding agents linked to first oligonucleotide probes, analyte detection reagents including analyte binding agents linked to second oligonucleotide probes, optional splint oligonucleotide probes complementary to portions of the first and second probes, and optional reagents such as lysis buffers, DNA ligase, DNA polymerase, dNTPs, and amplification buffers. One or more of the capture reagent, detection reagent, or lysis buffer comprises a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54). These kits support proximity-based ligation and amplification schemes for sensitive detection of target analytes.

[0001] The disclosure further encompasses machine-learning-enabled methods and systems for characterizing and optimizing surfactant performance in permeabilization workflows. In one embodiment, a biological particle (e.g., cell or other biological particle) is contacted with a permeabilization buffer comprising a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54), and the particle is analyzed with an analyzer (e.g., an instrument capable of detecting fluorescence emission and / or light scattering) to detect signals indicative of permeabilization or target labeling. Data generated from the analyzer are provided to a computing device, which applies a machine-learning process to determine one or more attributes associated with the performance of the surfactant or permeabilization buffer, such as permeabilization efficiency, intracellular target accessibility, surfactant concentration suitability, lot-to-lot consistency, or buffer stability. In some embodiments, data are collected from samples treated with permeabilizationbuffers containing multiple surfactant concentrations, and the machine-learning process recommends optimal surfactant concentrations or incubation conditions. Extracted features used for training or inference can include signal intensity, signal-to-background ratio, cell morphology, cell viability, fraction of labeled cells, and distributions of intracellular marker intensity. A system is provided comprising an analyzer and a computing device configured to implement these machine-learning processes and optionally output recommended parameters for subsequent assays.

[0021] In another aspect, compositions and methods are provided for stabilizing polypeptides. Compositions comprise a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54), at least one polar protic solvent, optional inorganic salts, optional buffering agents, optional chaotropic agents, a buffering agent, and one or more polypeptides. The compositions can be formulated as polypeptide storage buffers, polypeptide reaction buffers, immunoassay reaction buffers, enzymatic reaction buffers, master mixes, running buffers, or chromatography elution buffers. They may consist essentially of a buffering agent, polypeptide, and optionally salts, reducing agents, chelating agents, cryoprotectants, stabilizers, and / or carriers. The polypeptides include enzymes (e.g., nucleases, polymerases, reverse transcriptases, ligases, glycosylases, nuclease inhibitors, alkaline phosphatases, isomerases, transferases, oxidoreductases, lyases), growth factors, cytokines, matrix proteins, antibodies or fragments thereof, and antigens. The surfactant is present in concentrations typically from about 0.0001% to 10% (v / v). Methods are provided for stabilizing polypeptides by combining them with a surfactant-containing buffer to form a composition, which may optionally be lyophilized or airdried. Kits comprising such polypeptide-stabilizing compositions are also provided.

[0022] The surfactant-containing compositions and methods are additionally applied to viral inactivation and viral contamination control. Compositions are provided that comprise a surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54), in an amount effective to inactivate viruses, together with virus-containing materials such as Xenotropic Murine Leukemia Virus (XMuLV), Vesicular Stomatitis Virus (VSV), Adenovirus type 5 (AdV5), influenza virus, Hepatitis B virus (HBV), Herpes Simplex Virus (HSV), Cytomegalovirus (CMV), Human Immunodeficiency Virus (HIV), West Nile virus, Dengue virus, Zika virus, SARS-CoV-2, Ebolavirus, and others. The virus-containing compositions can include biopharmaceutical process streams, such as cell culture harvests, clarified harvests, chromatography eluates, filtration retentates, UF / DF fractions,recombinant protein or monoclonal antibody intermediates, plasma-derived intermediates, plasma fractions, vaccine bulks, viral seed train materials, and viral propagation mixtures. In some embodiments, the compositions include plasma-derived materials such as immunoglobulin, albumin, coagulation factors, or antithrombin. The surfactant is typically present at about 0.01% to 5% (w / v). Methods for inactivating viruses involve contacting viruses or virus-containing compositions with the surfactant under conditions sufficient to inactivate the virus, optionally followed by removal or dilution of the surfactant to a concentration below 0.01% (w / v) prior to downstream purification. Related methods are provided for reducing viral contamination in biological or biopharmaceutical compositions, and for using the surfactant in the manufacture of compositions for viral inactivation or for reducing viral contamination in recombinant protein or monoclonal antibody production.

[0023] The disclosure also provides surfactant-containing compositions and devices for lateral flow assays and capillary-based formats. A running buffer is provided that includes a surfactant according to Formula I (and / or related structures including any one of Formulas I A— I K and / or any one of the currently defined compounds 1-54), a polar protic solvent, and optional salts, buffering agents, and chaotropic agents, in an amount effective to improve capillary flow of sample through a lateral flow strip. A sample application pad for lateral flow assay devices is provided, comprising a porous substrate and a treatment composition deposited on or within the substrate. The treatment composition includes a nonionic surfactant according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54), at least one polar protic solvent, optional inorganic salts, buffering agents, and chaotropic agents, in an amount effective to enhance sample wetting and promote release of target analytes from biological samples. The treatment composition can further include buffers, blocking agents, and stabilizers. Lateral flow assay devices are disclosed that incorporate such sample pads, along with conjugate pads containing dried detection reagents (labeled analyte binding agents), porous membranes with test and control zones, downstream absorbent pads, and housings configured to support these components in a linear flow path. Upon sample application, the treated sample pad improves capillary flow and sample delivery, enabling generation of visually or instrumentally detectable signals indicative of the presence of the target analyte. In addition, surfactants according to Formula I (and / or related structures including any one of Formulas IA-IK and / or any one of the currently defined compounds 1-54) are used in the manufacture of lateral flow assay devices and strips.

[0024] Methods and kits are provided for determining LNP encapsulation efficiency. A method of the disclosure involves determining the amount of free mRNA in a composition containing LNPs, contacting the LNPs with a surfactant according to Formula I in an amount sufficient to release all mRNA, determining the total mRNA, and calculating the percentage of encapsulated mRNA. Kits for determining LNP encapsulation efficiency comprise a lysis buffer containing a surfactant according to Formula I (and / or related structures including any one of Formulas I A— I K and / or any one of the currently defined compounds 1-54), formulated as described above.

[0025] Throughout the disclosure, the surfactant-containing buffers and compositions can employ a variety of buffering systems, including citrate-based buffers (e.g., saline-sodium citrate), phosphate-based buffers (e.g., phosphate-buffered saline), Tris-based buffers, TAPS-based buffers, HEPES-based buffers, bicine-based buffers, tricine-based buffers, TAPSO-based buffers, TES-based buffers, MES-based buffers, PIPES-based buffers, cacodylate-based buffers, and combinations thereof. Detectable labels useful in the methods and kits include radiolabels, a wide range of organic and inorganic fluorophores and fluorescent constructs (e.g., fluoresceins, rhodamines, xanthene dyes, coumarins, cyanines, phycoerythrin and its conjugates, allophycocyanin and its conjugates, nanocrystals, Pdots, fluorescent conjugated polymers, and oligonucleotide-based fluorescent dyes), chromogenic substrates (e.g., diaminobenzidine, Fast Red, Fast Blue), haptens (e.g., fluorescein, biotin, DNP, digoxigenin), enzymatic labels (e.g., peroxidases, phosphatases, glycosidases, oxidases), detectable substrates for such enzymes (including horseradish peroxidase substrates and tyramides), and colorimetric protein or peptide detection dyes (e.g., dyes based on bicinchoninic acid and related chemistries).

[0026] Collectively, the disclosed surfactant compounds, compositions, methods, devices, and kits provide a versatile platform for cell permeabilization, intracellular and extracellular analyte detection, buffer and reagent formulation, reduction of non-specific binding in assays, improvement of capillary flow in lateral flow devices, stabilization of polypeptides, assessment of LNP encapsulation efficiency, and viral inactivation or viral risk reduction in biological and biopharmaceutical settings.

[0027] The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1A and 1 B are graphs showing results from using a compound according to the present disclosure (namely, Compound 1 (SY-2)) as a surfactant in cell lysing, wherein FIG.1 A shows a graph of results from a bicinchoninic acid (BCA) assay, wherein protein recovery was assessed for Compound 1 (SY-2), at various concentrations, as compared to the conventional non-ionic surfactants NP40 and EH9; and FIG. 1B shows is a graph illustrating the background signal from a mixture containing only the disclosed compound, buffer and a fluorescent dye, that is without a protein sample or cell lysate present.

[0029] FIG. 2 shows microscopic digital images of the cells after lysing treatment with compound 1 (SY-2), establishing that Compound 1 (SY-2) is sufficiently mild and does not lyse the nucleus.

[0030] FIG. 3 is a digital image showing a gel after performing SDS-PAGE gel electrophoresis and Coomassie staining on the lysate obtained from using the Compound 1 (SY-2) for cell lysis, and comparing with results obtained from using conventional non-ionic surfactants Tergitol™ NP-40 and EH9.

[0031] FIG. 4 is a digital image of a nitrocellulose membrane after transferring an SDS-PAGE gel run with the Compound 1 (SY-2), Tergitol™ NP-40, and EH9, and performing detection with EGFR and Cav1, wherein results are shown for the Compound 1 (SY-2), Tergitol™ NP-40, and EH9.

[0032] FIG. 5 is a digital image of a nitrocellulose membrane after transferring an SDS-PAGE gel run with the Compound 1 (SY-2), Tergitol™ NP-40, and EH9, and performing detection with ATP1A1 and COXIV, wherein results are shown for the Compound 1 (SY-2), Tergitol™ NP-40, and EH9.

[0033] FIG. 6 shows representative images of cells permeabilized with SY-No-dPEG9-OMe and visualized with anti-a-tubulin antibody (1:500).

[0034] FIG. 7 shows representative images of cells permeabilized with SY-DMB-dPEG9-OMe and visualized with anti-TGN46 antibody, rhodamine-phalloidin, and DAPI, along with along with composite images with and without primary antibody.

[0035] FIG. 8 shows representative images of cells permeabilized with SY-iPe-PEG9-OMe and visualized with anti-mitofilin antibody, rhodamine-phalloidin, and DAPI along with composite images with and without primary antibody.

[0036] FIG. 9 shows representative images of cells permeabilized with SY-DMB-dPEG9-OMe and visualized with anti-pericentrin antibody, rhodamine-phalloidin, and DAPI, along with composite images with and without primary antibody.

[0037] FIG. 10 shows representative images of cells permeabilized with SY-DMB-dPEG9-OMe and visualized with anti-histone H3 antibody, rhodamine-phalloidin and DAPI, along with composite images with and without primary antibody.

[0038] FIG. 11 A shows flow cytometry spectra of 1 pg / test of antibody in permeabilization buffer containing 0.1% or 0.2% of SY-DMB-dPEG9-OMe and SY-iPe-dPEG9-OMe compared to 0.1% or 0.2% saponin.

[0039] FIG. 11 B shows flow cytometry spectra of 1 pg / test of antibody in permeabilization buffer containing 0.1% or 0.2% of SY-Oc-dPEG9-OMe, SY-Pe-dPEG9-OMe or SY-No-dPEG9-OMe.

[0040] FIG. 12 is a bar graph, illustrating the staining index with Control Permeabilization Buffer compared with several exemplary Permeabilization Buffers of the present disclosure comprising Syringic Acid based compounds.

[0041] FIG 13 illustrates an example graphical user interface (GUI) configured for use in performing some or all of the computational methods described herein.

[0042] FIG 14 is a block diagram of an example computing device configured to perform some or all of the computational methods described herein.

[0043] FIG. 15 is a block diagram of an example scientific instrument system in which some or all of the computational methods described herein may be implemented.DETAILED DESCRIPTIONTerms and Definitions

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of microscopy, biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, bioprocessing, pharmaceutical, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0045] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open-ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed disclosure. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0046] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified. As used herein, the term “or” can be conjunctive or disjunctive. As used herein, the term “and / or” refers to both the conjunctive and disjunctive. As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0047] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” may refer to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” may mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbolmeans “about” or “approximately.”

[0048] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1 -2.0 includes 0.1, 0.2, 0.3, 0.4... 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0049] As used herein, “protein," “peptide,” and ’’polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.

[0050] The term "antibody" as used herein refers to a polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen. Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VHregion and the VLregion are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Some mammals, such as camels, alpacas, and llamas, have heavy-chain antibodies that lack a light chain. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles, and has some functionality similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish such as sharks, while IgX antibodies are found in amphibians. IgNAR antibodies are heavy-chain antibodies.

[0051] Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U. S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901 -917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40: W521 -524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online.

[0052] A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domainantibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks, which produce heavy-chain antibodies (IgNARs). Camelid VHH antibodies are produced by several species including camel, llama, alpaca, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.

[0053] The term “enzyme” as used herein refers to any biological macromolecule, for example a protein, that acts as a biological catalyst, accelerating chemical reactions. The term “antigen” refers to a molecule, or portion thereof, that can bind to a specific antibody or T-cell receptor. The term “antigen” also refers to a compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. Exemplary antigens include, but are not limited to, dsDNA, ssDNA, histones (such as H1, H2A, H2B, H3, H4), nucleosomes, RNP / Sm, Sm (B / B’, D), 111 -RNP, SS-A / Ro52, SS-A / R06O, SS-B / La, Scl-70 I Topoisomerase I, Jo-1 (Histidyl-tRNA synthetase), PL-7, PL-12, EJ, OJ, PM / Scl-75, PM / Scl-100, U3-RNP / Fibrillarin, Ku, Mi-2, SRP (Signal Recognition Particle), CENP-A, CENP-B, CENP-C, Thyroglobulin (Tg), Thyroid Peroxidase (TPO), TSH Receptor (TSHR) fragments or extracellular domain, CCP (Cyclic Citrullinated Peptides), citrullinated vimentin, RF (IgG Fc fragments), Proteinase 3 (PR3), Myeloperoxidase (MPO), GAD65 (Glutamic Acid Decarboxylase 65), IA-2 / ICA512, ZnT8 (Zinc transporters), Insulin / Proinsulin, Tissue Transglutaminase (tTG), Deamidated Gliadin Peptides (DGP), Myelin Basic Protein (MBP), MOG (Myelin Oligodendrocyte Glycoprotein), Acetylcholine Receptor (AChR) extracellular domain, MuSK (Muscle-specific kinase), Hu (ANNA-1), Ri (ANNA-2), Yo (PCA-1), CV2 / CRMP5, Amphiphysin, Desmoglein 1, Desmoglein 3, BP180 (Collagen XVII), BP230, Type VII Collagen (EBA), Mitochondrial M2 antigens (E2 subunit of PDC), LKM-1 (CYP2D6), SLA / LP (Soluble Liver Antigen), ASCA antigens (mannan from Saccharomycescerevisiae), IL-24, elF6,, Rheumatoid Factor, Jo-1 (Histidyl-tRNA synthetase), PL-7 tRNA synthetase, PL-12 tRNA synthetase, EJ tRNA synthetase, OJ tRNA synthetase, Mi-2, TIF1 -y, MDA5, NXP-2, SAE, and Intrinsic Factor (pernicious anemia).

[0054] As used herein, the terms “stabilization,” “stabilizing,” and “stabilized,” when used in reference to enzyme activity refer to the ability of a material to maintain, enhance, or otherwise inhibit the decline or loss of the activity of an enzyme, often as measured over time (i.e., in the presence of a stabilizer, an enzyme retains its activity for a longer time period than the enzyme in the absence of the stabilizer). An exemplary stabilizer is BSA. “Stabilization of enzyme activity” also refers to the ability of a material to maintain the activity of an enzyme under suboptimalconditions of temperature or pH. As another example, “stabilizing enzyme activity” refers to the ability of a material to enhance enzyme activity under suboptimal conditions, as compared to activity in the absence of a “stabilizing” compound or material.

[0055] As used herein, the term “carrier” refers to any material, substance, matrix, vehicle, excipient, or support that facilitates the delivery, stabilization, handling, immobilization, transport, dispersion, or presentation of one or more active, functional, or detectable components. An example of a carrier is BSA.

[0056] As used herein, the term “cryoprotectant” refers to any compound, mixture, or formulation that mitigates or prevents damage to biological or chemical materials during exposure to low temperatures, freezing, or cryogenic storage. A cryoprotectant may function by reducing ice crystal formation, stabilizing macromolecular structures, maintaining osmotic balance, preserving membrane integrity, or otherwise enhancing the viability, functionality, or structural preservation of cells, proteins, nucleic acids, reagents, or other materials subject to freezing. Cryoprotectants may be permeating or non-permeating and may include, without limitation, sugars, polyols, amino acids, polymers, salts, glycerol, dimethyl sulfoxide (DMSO), antifreeze proteins, cryoprotective agents (CPAs), or combinations thereof. In certain embodiments, the cryoprotectant is supplied as a component of a buffer, medium, formulation, or storage solution.

[0057] As used herein, the term “buffering agent” refers to a compound or mixture that helps maintain a stable pH in a solution. A buffering agent may include, but is not limited to any of MOPS (3-(N-morpholino)propanesulfonic acid), citrate buffers (saline-sodium citrate (SSC)), phosphate buffers (phosphate buffered saline (PBS)), tris (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-1,3-diol)-based buffers, TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid)-based buffers, HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid)-based buffers, Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid)-based buffers, tricine (N-[tris(hydroxymethyl)methyl]glycine)-based buffers, TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)-based buffers, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)-based buffers, MES (2-(N-morpholino)ethanesulfonic acid)-based buffers, PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid))-based buffers, Cacodylate (dimethylarsenic acid)-based buffers, or any combination thereof.

[0058] The term “storage buffer” refers to a buffering solution in which a polypeptide (e.g. an enzyme or antibody) is stored.

[0059] As used herein, the term “reaction buffer” refers to a buffering solution which provides physicochemical conditions that support the catalytic activity, stability, and specificity of one or more polypeptides or nucleic acids during a reaction.

[0060] The terms “chelator” or “chelating agent” refer to any materials having more than one atom with a lone pair of electrons that are available to bond to a metal ion. Exemplary chelators useful in the present disclosure include, but are not limited to, EDTA (ethylenediaminetetraacetic acid), Citrate, DTPA, DTT, EGTA, transferrin, and the like. The chelator is preferably EDTA.

[0061] The term “reducing agent” refers to material that donates electrons to a second material to reduce the oxidation state of one or more of the second material's atoms, and to break disulfide bonds, which is crucial for protein analysis and purification. Exemplary reducing agents include, but are not limited to, EDTA, tris (2-carboxyethyl) phosphine hydrochloride (TCEP), p-mercaptoethanol (BME), dithiothreitol (DTT), Ellman's Reagent, and Hydroxylamine-HCI and the like. The reducing agent is preferably DTT.

[0062] As used herein, the term “analyte binding agent” refers to a molecule or moiety capable of binding to a macromolecular constituent (e.g., an analyte, e.g., a biological analyte). Analyte binding agents provided herein can include, but are not limited to, an antibody, or an epitope binding fragment thereof, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The analyte binding agent can specifically bind to a target analyte with high affinity and / or with high specificity. Exemplary analyte binding agents can also include a nucleotide sequence (e.g., an oligonucleotide), which can correspond to at least a portion or an entirety of the analyte binding agent. The analyte binding agent can include a polypeptide and / or an aptamer (e.g., a polypeptide and / or an aptamer that binds to a specific target molecule). The analyte binding agent can include an antibody or antibody fragment (e.g., an epitope-binding fragment) that binds to a specific target (e.g., a polypeptide). Analyte binding agents can include or be conjugated to detectable labels, solid supports (e.g., beads, particles, matrices, filters, and the like). Analyte binding agents include “capture antibody reagents” and “detection antibody reagents.”

[0063] Target analytes can be proteins (e.g., a protein or polypeptide on a surface of the biological sample (e.g., a cell) or an intracellular or nuclear protein or polypeptide), carbohydrates, nucleic acids (e.g., DNA or RNA) lipids, or the like. Analyte binding agents can beprovided as a plurality of analyte binding agents wherein different analyte binding agents specifically bind to different target analytes or different epitopes on a single target analyte. Alternatively, a plurality of analyte binding agents may specifically bind to single species of analyte (e.g., a single species of polypeptide). For example, the plurality of analyte binding agents can be a plurality of the same analyte binding agent. Where the plurality of analyte binding agents specifically bind to a single species of a target analyte, the analyte binding moieties can bind to different epitopes of the target analyte, or a different form of the analyte (e.g., a phosphorylated form of the analyte or the unphosphorylated form of the analyte). In some embodiments, the plurality of analytes includes multiple different species of analyte (e.g., multiple different species of polypeptides).

[0064] As used herein, the term “detectable signal” or “detectable label” refers to a chemical, biological, or physical marker attached to a molecule that produces a measurable signal— including but not limited to, fluorescence, color, radioactivity, light, or mass — allowing the detection, quantification and / or characterization of the molecule to which it is bound. Detectable labels that may be used in various embodiments disclosed herein include, but are not limited to, radiolabels, organic fluorophores, fluorescent proteins, quantum dots, chromophores, enzymatic labels, chemiluminescent labels, bioluminescent labels, metal-based labels, oligonucleotide-based labels, affinity tags, haptens, detectable substrates, colorimetric proteins, peptide detection dyes, or any combination thereof. Exemplary detectable labels include, but are not limited to ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, nanoparticles (e.g., a gold nanoparticle), enzymes, colorimetric labels, magnetic labels, small molecules (e.g., biotin), streptavidin, haptens, allophycocyanin (APC), oligonucleotides, and peptide tags (e.g., Myc tag, His tag, or FLAG tag). Chromophores include 3,3'-diaminobenzidine, 4-chloro-2-methylbenzenediazonium (Fast Red), 3,3'-dimethoxybiphenyl-4,4'-di(diazonium) or zinc chloride (Fast Blue). Haptens include fluorescein, biotin, nitroaryls, dinitrophenol (DNP), digoxigenin, oxazole, pyrazole, thiazole, benzofuran, urea, thiourea, rotenoid, coumarin, or cyclolignan. Enzymatic labels include peroxidase, a phosphatase, a glycosidase, or an oxidase. Detectable substrates include a substrate for a peroxidase, a substrate for horseradish peroxidase, a tyramide, or atyramide-like molecule. Colorimetric protein or peptide detection dye include bicinchoninic acid (BCA), bathocuprione, bathocuprionedisulphonic acid, tartarate, copper sulphate, acetonitrile, salts thereof, derivatives thereof, and combinations thereof.

[0065] Labels may be molecules (either attached to analyte binding agents or not) to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Fluorescent labelsare also referred to as fluorophores, fluorescent tags, fluorescent dyes, or fluorescent probes. A fluorescent label may be a naturally occurring fluorescent protein (e.g., phycoerythrin, PE), a derivative thereof (e.g., PE-Cy7), a tandem dye, a polymer dye, a single molecule dye, an organic dye, a fluorescent nucleic acid, a fold-back oligonucleotide probe with a complementary 3' end for fluorescent dye incorporation, or a scaffold-based fluorescent label, for example a nucleic acid nanostructure including fluorescent DNA nanostructures such as PHTION nucleic acid nanostructures, including NOVAFLUOR dyes (Thermo Fisher Scientific, Waltham, MA). Other fluorophore labels that may be used include xanthenes, fluoresceins, rhodamines, rhodols, roseamines, carbopyranonse,indoles, indacenes, borapolyazaindacenes, furans,benzofurans, cyanines, benzocyanines, benzopyriliums, pyrenes, coumarins, carbostyryls, styryls, squarines, resorufins, anthraquinones, acridines, benzophenoxazines, cyanine-based tandem dyes, phycoerythrin-dye conjugates, allophycocyanins, allophycocyanin-dye conjugates, nanocrystals, Pdots, fluorescent conjugated polymers, and oligonucleotide-based fluorescent dyes.

[0066] As used herein, the term “sample” or “biological sample” generally refers to a collection of cells or to a tissue. Generally, a tissue contains multiple cells, often similar cells that may perform the same or similar functions. The sample may be a cell sample. The sample may be acell line orcell culture sample. The sample may be an environmental sample, soil, sediment, dust, air particulates, surface swabs, or water samples from any natural or artificial source. The sample can include one or more cells, or one or more cell aggregates or clusters. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be whole blood, plasma, or serum. The sample may be a tissue from a diseased or cancerous organ, or one suspected of being diseased or cancerous. Example tissue types in animals may include connective, epithelial, brain, adipose, muscle and nervous tissue. The sample may be a fluid sample, such as a blood sample, urine sample, buccal sample, cerebrospinal fluid sample, amniotic fluid sample, semen sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. In some examples, a sample may comprise any number of macromolecules, for example, cellular macromolecules or cellular analytes. The sample may be a stool sample. The sample may be a Formalin-Fixed Paraffin-Embedded (FFPE) tissue sample, fresh-frozen tissue, fresh (unfixed) tissue, cryopreserved tissue, OCT-embedded tissue (optimal cutting temperature compound), microdissected tissue (e.g., lasercapture microdissected (LCM) tissue), or a cytology sample. The present disclosure is not limited to any particular type of tissue or sample.

[0067] As used herein, the term “immunoassay” refers to any one of a number of analytical methods that detect, quantify, or characterize an analyte through a specific binding interaction between an antibody (or antibody fragment) and the antigen or analyte to which it is bound.

[0068] As used herein, the term “master mix” may be defined as pre-formulated composition with all or many of the essential reaction components except for the sample to be analyzed (e.g., nucleic acid template, primers, probes, nucleotides, enzymes, reaction buffers, or other reactants depending on the specific master mix). A “PCR master-mix,” for example, can be a pre-formulated composition with all or many of the essential reaction components except for the sample to be analyzed by a polymerase chain reaction (PCR) amplification.

[0069] As used herein, “PAGE” refers to polyacrylamide gel electrophoresis. “SDS-PAGE” refers to sodium dodecyl sulfate (“SDS”) polyacrylamide gel electrophoresis.

[0070] As used herein “polyacrylamide” refers to a mixture of acrylamide (CH2=CHC(O)NH2) and bisacrylamide (CH2[NHC(O)CH=CH2]2). The percentage of polyacrylamide in the gel casting solution determines the gel porosity, with typical percentages ranging from 5-25%. The molecular weight of the biomolecule determiners the percentage of the gel, with greater molecular weights requiring lower percentage gels for optimal resolution.

[0071] An “electrophoresis gel casting solution” as described herein refers to the solution used to cast an electrophoretic gel. The solution contains a percentage of polyacrylamide, a buffer, and water. For SDS-PAGE, the electrophoresis gel casting solution typically contains 8-12.5% polyacrylamide, Tris-glycine buffer, and sodium dodecyl sulfate (“SDS”). The solution is cast by adding a polymerization initiator, such as the oxidizing agent ammonium persulfate (APS). Often a polymerization accelerator such as tetramethylethylenediamine (TEMED) is added to facilitate rapid polymerization. Upon adding the polymerization initiator, the gel polymerizes within 10-30 min. However, the use of other casting solutions and buffers is also contemplated.

[0072] As used herein, a “stacking gel” refers to an electrophoretic gel that comprises at least two layers - an upper gel having a lower pH and lower percentage of polyacrylamide, and a lower gel, or “resolving gel” that has a greater pH and higher percentage of polyacrylamide. The stacking gel permits the sample volume in the well to be concentrated so that the entire sample enters the resolving gel in a focused, narrow band.

[0073] As used herein, a “running buffer” refers to the buffers used in various methods such as gel electrophoresis, column chromatography, lateral flow assays and the like. Typical SDS-PAGE running buffer (1 x concentration) consists of 25 mM Tris hydroxy amino methane (Tris), 192 mM glycine, pH 8.3, and 0.1% sodium dodecyl sulfate (SDS). Chromatography running buffers (sometimes referred to as mobile phase buffers) refer to the mobile-phase solution that maintains pH and other chemical conditions required for effective separation of molecules as they move though a chromatographic column or matrix. Typical chromatography running buffers include buffering agents, organic solvents, and the like. Lateral flow assay running buffers can also be referred to as an “assay buffer” or a “chase buffer,” and promote consistent capillary flow along the lateral flow assay strip, while reducing non-specific binding and background, and stabilizing proteins, antibodies and detectable labels. Lateral flow assay running buffers may include surfactants, and one or more of a buffering agent, a salt, a blocking agent, a preservative, or a crowding agent. However, the use of a variety of running buffers and gel chemistries is contemplated and the description here is a mere example. Surfactants may be present in any of the running buffers provided herein.

[0074] As used herein, an “elution buffer” refers to an aqueous composition formulated to release, displace, or otherwise separate a target analyte from a chromatographic stationary phase. The elution buffer may effect such release by altering one or more physicochemical conditions — such as ionic strength, pH, solvent composition, chaotropic strength, or specific ligand competitors — to reduce or eliminate interactions between the analyte and the stationary phase. In certain embodiments, the elution buffer comprises salts, pH-adjusting agents, organic solvents, surfactants, chelators, reducing agents, stabilizers, or other functional components that promote selective recovery of bound molecules. The elution buffer may be applied in a stepwise, gradient, or continuous format and is suitable for use in affinity, ion-exchange, hydrophobic interaction, mixed-mode, reverse-phase, or other chromatographic systems.

[0075] As used herein, a “blocking buffer” refers to a composition comprising one or more blocking agents that, when contacted with an assay component (including but not limited to a solid support (e.g., a reaction vessel, membrane, microplate well, bead, sensor surface, or other assay substrate)), reduces or prevents non-specific binding or adsorption of assay reagents and / or analytes to that component. Blocking buffers may also be used to inhibit non-specific interactions between polypeptides while maintaining specific binding between analytes and analyte binding agents. Blocking buffers may, for example, include one or more blocking agents, including serum albumin, casein, non-fat dry milk, milk proteins, bovine serum albumin (BSA) fetal calf serum(FCS), gelatin, peptides, polymers, surfactants, or other excipients, and may further comprise salts, buffering agents, preservatives, stabilizers, or other optional components, provided that the composition is effective to occupy or passivate non-specific binding sites without substantially interfering with the specific binding interaction(s) measured in the assay (e.g., immunoassays, nucleic acid hybridization assays, enzyme assays, or ligand-receptor assays).

[0076] As used herein, an “electrophoretic gel running apparatus” refers to an assembly comprising at least one electrophoretic gel assembly, a negative terminal, a positive terminal a buffer reservoir at the positive and negative terminals, and a power supply for providing electric current. Typical electrophoretic gels are run with the negative terminal proximate to the wells (top) and the positive terminal proximate to the opposite side (bottom). The electrophoretic gel assembly comprises a gel cassette typically comprises two plates: a retainer plate and a divider plate coupled to form a cavity between the retainer plate and the divider plate into which a gel casting solution is poured. A comb is used to create sample holder wells in the gel. Gels can be pre-cast gels or self pour gels. Many formats of electrophoresis gel assemblies are known and can be used with the formulations, kits, and methods of the present disclosure

[0077] As used herein, a “mountant” refers to any composition, medium, or formulation that is applied to, contacted with, or used to support a biological, chemical, or synthetic sample for the purpose of facilitating observation, imaging, handling, preservation, stabilization, storage, or analysis of the sample.

[0078] As used herein, the term “virus-containing composition” generally refers to a composition (solution, suspension, or mixture) that comprises one or more viruses or is suspected of containing viruses. For example, a biological or biopharmaceutical material in which viral contamination may be present and from which viruses need to be inactivated or eliminated. Further examples include cell culture supernatants from bioreactors (harvest fluids), purified or partially purified protein solutions, blood products, vaccines, or any matrix carrying viral particles. Biopharmaceutical process intermediates are often virus-containing compositions, i.e., an immunoglobulin solution or plasma protein fraction that might harbor adventitious viruses, or a recombinant protein harvest from cell culture that could contain retroviral particles. A viruscontaining composition may be a liquid solution (e.g. an aqueous buffer solution containing the product of interest and contaminants) or a semi-liquid preparation, and it can derive from human / animal sources or from cell culture. Viruses in such a composition can be an enveloped or non-enveloped virus that is targeted for inactivation. It is understood that virus-containing composition may be a medium to be treated in the claimed method, which contains a virus and may be subjected to treatment with compounds of the present disclosure.

[0079] As used herein, the term “plasma-derived product” generally refers to a biological product derived from human or animal blood plasma, especially therapeutic proteins obtained by fractionating blood plasma. Plasma-derived products (also called plasma-derived medicinal products) include, for example, immunoglobulin preparations (IVIG or subcutaneous IG), coagulation factor concentrates (such as Factor VIII or Factor IX for hemophilia), human albumin solutions, fibrinogen, protease inhibitors (e.g., antithrombin III), and other proteins isolated from plasma. These products may be manufactured via large-scale plasma pooling and fractionation processes and undergo virus inactivation steps as part of ensuring pathogen safety. A plasma-derived product may be virus-containing composition. It is appreciated that a plasma-derived product may be treated by the claimed method (adding a compound of this disclosure) to inactivate any viruses that might be present. Plasma-derived products may comprise a licensed therapeutic product or intermediate obtained from blood plasma, which by its nature could carry blood-borne viruses and hence is subjected to viral clearance (i.e., surfactant-mediated virus inactivation) processing as described herein.

[0080] As used herein, the term “surfactant-mediated virus inactivation” generally refers to a virus inactivation process that is carried out by adding a surfactant to a virus-containing composition, under conditions that allow the surfactant to disrupt or deactivate the viruses. In biopharmaceutical manufacturing, surfactant-mediated virus inactivation is a well-established method for eliminating lipid-enveloped viruses: the surfactant inserts into or solubilizes the lipid envelopes, effectively destroying the effectivity of the virus. For example, solvent / surfactant treatment used in plasma product manufacturing, where a nonionic surfactant as described herein may be combined with a solvent such as tri-n-butyl phosphate and is incubated with the plasma fraction to inactivate HIV, HBV, HCV, and other enveloped viruses. Surf actant- mediated virus inactivation may be an any virus-killing method that employs a surfactant as the active agent. In the context of the claims, the compounds of this disclosure act as the surfactant accomplishing virus inactivation, i.e., disruption of viral membranes or capsids by the amphiphilic molecules. This process is typically performed in solution, at a controlled temperature and for a specified time (for example, treatment of a protein solution with 1% surfactant for 60 minutes at 25-30 °C). The outcome of surfactant-mediated inactivation is that the targeted viruses are rendered non-infectious (unable to replicate in a host) by the action of the surfactant. Surfactant-mediated inactivation may be more effective for enveloped viruses, since the surfactant attacks the lipid envelope, but certain formulations and conditions can also reduce infectivity of some non-enveloped viruses to a degree.

[0081] As used herein, the term “sufficient to inactivate the virus” generally refers to a set of treatment parameters (such as surfactant concentration, contact time, temperature, and any other relevant conditions) that, taken together, achieve the desired virus inactivation in the method. After treating the composition under those conditions, the virus is effectively inactivated, i.e., no longer capable of causing infection. It is acknowledged that industry standards (for example, achieving at least a 4 log₁₀ reduction in viral titer is a common benchmark of effective inactivation) would be appliable with respect to these conditions such that these conditions can vary depending on the specific virus and composition, but generally include using an adequate amount of a compound of this disclosure (for instance, 0.3% to 1% by volume, or a concentration above the critical micelle concentration of the surfactant), allowing it to incubate for a sufficient time (e.g., 30 minutes, 60 minutes, or longer), and maintaining a suitable temperature (often ambient room temperature -20-30 °C, or slightly elevated, to facilitate the reaction). Inactivation of the virus can be confirmed by standard infectivity assays, i.e., cell culture assays showing no viral growth or a reduction below detection limits. Conditions sufficient to inactivate the virus may therefore refer to effective treatment conditions that result in the virus being non-infectious. In one exemplary set of conditions is incubating a virus-spiked sample with 1% of a compound of this disclosure at 25 °C for 60 minutes, which has been shown to produce significant viral titer reductions (e.g., >4 log₁₀ reduction for enveloped retrovirus in a model system). Another example of sufficient conditions could be gently agitating a plasma product with 0.5% surfactant at 30 °C for 2 hours.

[0082] As used herein, the term “Log Reduction Factor (LRF)” generally refers to a standard quantitative measure of how effectively a virus inactivation method reduces viral infectivity. The higher LRF the more effective virus inactivation. As noted above, achieving at least a 4 log₁₀ reduction in viral titer is a common benchmark of effective inactivation. LRF is used in biologies and plasma product manufacturing to validate viral clearance processes, whereby LRF is a function of viral titer, which is typically measured in TCID50 / mL (median tissue culture infectious dose) or PFU / mL (plaque-forming units). LRF is calculated as below:viral titer fbefere tres ti X'■ I. mid vsrai titer ■ after treatment > /

[0083] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure which are, for brevity,described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0084] Although the steps of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, steps described sequentially may in some cases be rearranged or performed concurrently. Additionally, the description sometimes uses terms like “produce” or “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual steps that are performed. The actual steps that correspond to these terms will vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.

[0085] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.

[0086] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and / or limits of detection under standard test conditions / methods and in some aspects encompasses a range up to ± 15% of that numerical value, unless the context clearly dictates otherwise.

[0087] Certain functional group terms used herein include a symbol which is used to show how the defined functional group attaches to, or within, the compound to which it is bound. Also, a dashed bond (i.e., “ — ”) as used in certain formulas described herein indicates an “optional” bond to a substituent or atom of the formula other than hydrogen in the sense that the bond (and in some embodiments, the substituent) may or may not be present. In any formulas comprising a dashed bond, if the optional bond and / or any corresponding substituent is notpresent, then the valency requirements of any atom(s) bound thereto is completed by a bond to a hydrogen atom.

[0088] The symbol ” is used to indicate a bond disconnection in abbreviated structures / formulas provided herein. A person of ordinary skill in the art recognizes that the definitions provided below and the compounds and formulas included herein are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. In formulas and compounds disclosed herein, a hydrogen atom is present and completes any formal valency requirements (but may not necessarily be illustrated) wherever afunctional group or other atom is not illustrated. For example, a phenyl ring that is drawn ascomprises a hydrogen atom attached to each carbon atom of the phenyl ring other than the “a” carbon, even though such hydrogen atoms are not illustrated. Any functional group disclosed herein and / or defined above can be substituted or unsubstituted, unless otherwise indicated herein.

[0089] If a group R is depicted as “floating” on a ring system, as for example in the group:then, unless otherwise defined, a substituent R can reside on any atom of the fused bicyclic ring system, excluding the atom carrying the bond with the” symbol, so long as a stable structure is formed. In the example depicted, the R group can reside on an atom in either the left side or the right side ring of the naphthylene ring system.

[0090] When there are more than one such depicted “floating” groups, as for example in the formula:where there are two groups, namely, the R and the bond indicating attachment to a parent structure; then, unless otherwise defined, the “floating” groups can reside on any atoms of the ring system, again assuming each replaces a depicted, implied, or expressly defined hydrogen on the ring system and a chemically stable compound would be formed by such an arrangement.

[0091] To facilitate review of the disclosure, the following explanations of specific terms are provided.

[0092] Aldehyde: -C(O)H.

[0093] Aliphatic: A hydrocarbon group having at least one carbon atom to 50 carbon atoms (C1-50), such as one to 25 carbon atoms (C1-25), or one to ten carbon atoms (CMO), and which includes alkanes (or alkyl), alkenes (or alkenyl), alkynes (or alkynyl), including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Aliphatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0094] Alkenyl: An unsaturated monovalent hydrocarbon having at least two carbon atoms to 50 carbon atoms (C2-50), such as two to 25 carbon atoms (C2-25), or two to ten carbon atoms (C2-10), and at least one carbon-carbon double bond, wherein the unsaturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent alkene. An alkenyl group can be branched, straight-chain, cyclic, cis, or trans (e.g., E or Z).

[0095] Alkoxy: -O-aliphatic, such as -O-alkyl, -O-alkenyl, -O-alkynyl; with exemplary embodiments including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, f-butoxy, sec-butoxy, n-pentoxy (wherein any of the aliphatic components of such groups can comprise no double or triple bonds, or can comprise one or more double and / or triple bonds). Alkoxy groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0096] Alkyl: A saturated monovalent hydrocarbon having at least one carbon atom to 50 carbon atoms (C1-50), such as one to 25 carbon atoms (C1-25), or one to ten carbon atoms (Ci-10), wherein the saturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent compound (e.g., alkane). An alkyl group can be branched, straight-chain, or cyclic.

[0097] Alkynyl: An unsaturated monovalent hydrocarbon having at least two carbon atoms to 50 carbon atoms (C2-50), such as two to 25 carbon atoms (C2-25), or two to ten carbon atoms (C2-10), and at least one carbon-carbon triple bond, wherein the unsaturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent alkyne. An alkynyl group can be branched, straight-chain, or cyclic.

[0098] Amide: -C(O)NRbRcor -NRbC(O)Rcwherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group and can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0099] Amino: -NRbRc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group, and can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0100] Aromatic: A cyclic, conjugated group or moiety of, unless specified otherwise, from 5 to 15 ring atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); that is, at least one ring, and optionally multiple condensed rings, have a continuous, delocalized iT-electron system. Typically, the number of out of plane n-electrons corresponds to the Huckel rule (4n + 2). The point of attachment to the parent structure typically is through an aromatic portion of thecondensed ring system. For example,0. However, in certain examples, context or express disclosure may indicate that the point of attachment is through a non-aromatic portion ofNthe condensed ring system. For example,. An aromatic group or moiety may comprise only carbon atoms in the ring, such as in an aryl group or moiety, or it may comprise one or more ring carbon atoms and one or more ring heteroatoms comprising a lone pair of electrons (e.g. S, O, N, P, or Si), such as in a heteroaryl group or moiety. Aromatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0101] Aroxy: -O-aromatic. Aroxy groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0102] Aryl: An aromatic carbocyclic group comprising at least five carbon atoms to 15 carbon atoms (C5 -15), such as five to ten carbon atoms (C5-10), having a single ring or multiple condensed rings, which condensed rings can or may not be aromatic provided that the point of attachment to a remaining position of the compounds disclosed herein is through an atom of the aromatic carbocyclic group. Aryl groups may be substituted with one or more groups other than hydrogen, such as an aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0103] Azo: -N=NRawherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Azo groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0104] Carbamate: -OC(O)NRbRc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Carbamate groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0105] Carbonate: -OC(O)ORa, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Carbonate groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. In independent embodiments, Racan be hydrogen.

[0106] Carboxyl: -C(O)OH.

[0107] Carboxylate: -C(O)O_or salts thereof, wherein the negative charge of the carboxylate group may be balanced with an M+counterion, wherein M+may be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4 where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]05, or [Ba2+]0.5.

[0108] Cyano: -CN.

[0109] Degree of Polymerization: The number of monomer units in a polymer. In the context of the present disclosure, when discussing, for example, polyalkene oxide and / orpolyalkylene amine polymers comprising repeat units of monomers, the degree of polymerization is typically defined by a mass average molecular weight.

[0110] Disulfide: -SSRa, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Disulfide groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0111] Dithiocarboxylic: -C(S)SRawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Dithiocarboxylic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0112] Ester: -C(O)ORaor -OC(O)Ra, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Ester groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0113] Ether: -aliphatic-O-aliphatic, -aliphatic-O-aromatic, -aromatic-O-aliphatic, or -aromatic-O-aromatic, including any polymers thereof having repeats of any such groups (e.g., polyalkene oxide compounds). Ether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0114] Halo (or halide or halogen): Fluoro, chloro, bromo, or iodo. In some embodiments, halo can also include astatine.

[0115] Haloaliphatic: An aliphatic group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such as fluoro, bromo, chloro, or iodo. Haloaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0116] Haloheteroaliphatic: A heteroaliphatic group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such as fluoro, bromo, chloro, or iodo. Haloheteroaliphatic groups can be substituted with one or moregroups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0117] Heteroatom: An atom other than carbon or hydrogen, such as (but not limited to) oxygen, nitrogen, sulfur, silicon, boron, selenium, or phosphorous. A heteroatom does not include a halogen atom.

[0118] Heteroaliphatic: An aliphatic group comprising at least one heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one to 5 heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorous, and oxidized forms thereof within the group. Alkoxy, ether, amino (excluding NH2), disulfide (wherein Rais other than H), peroxy (wherein Rais other than H), and thioether groups are exemplary (but non-limiting) examples of heteroaliphatic. Heteroaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0119] Hydrophilic: A hydrophilic group according to the present disclosure is a functional group or other chemical group that exhibits affinity for water. In some aspects, a hydrophilic group can further comprise a lipophilic portion; however, in such instances, the hydrophilic group is predominately hydrophilic and the lipophilic portion makes up a minor (e.g., less than 50%) of the hydrophilic group. Solely by way of example, a hydrophilic group can comprise a combination of a heteroaliphatic group and an aliphatic group and still be hydrophilic because the heteroaliphatic group makes up a greater proportion of the molecular weight of the total hydrophilic group than does the aliphatic group.

[0120] Hydrophilic-Lipophilic Balance (HLB): A numerical value that represents the balance of the size and strength of the hydrophilic and lipophilic moieties of a surfactant compound. The HLB scale ranges from 0 to 20.

[0121] Hydroxyl: -OH

[0122] Ketone: -C(O)Ra, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Ketone groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0123] Lipophilic: A lipophilic group according to the present disclosure is a functional group or other chemical group that lacks affinity for water.C9H19

[0124] Tergitol™ NP-40: A compound having a structure

[0125] Organic Functional Group: A functional group that may be provided by any combination of aliphatic, heteroaliphatic, aromatic, haloaliphatic, and / or haloheteroaliphatic groups, or that may be selected from, but not limited to, aldehyde; aroxy; acyl halide; halogen; nitro; cyano; azide; carboxyl (or carboxylate); amide; ketone; carbonate; imine; azo; carbamate; hydroxyl; thiol; sulfonyl (or sulfonate); oxime; ester; thiocyanate; thioketone; thiocarboxylic acid; thioester; dithiocarboxylic; phosphonate; phosphate; silyl ether; sulfinyl; sulfonamide; thial; or combinations thereof. Organic functional groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0126] Oxime: -CRa=NOH, wherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Oxime groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0127] Peroxy: -O-ORawherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Peroxy groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0128] Phosphate: -O-P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge, which can be balanced by a counterion, M+, wherein each M+independently can be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4 where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]05, or [Ba2+]o.5. The Ragroups of the phosphate can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0129] Phosphonate: -P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge, which can be balanced by a counterion, M+, wherein each M+independently can be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4 where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]o.5, or [Ba2+]0.5. The Ragroups of the phosphonate group can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0130] Silyl Ether: -OSiRaRbRc, wherein each of Ra, Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Silyl ether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0131] Sulfinyl: -S(O)Ra, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfinyl groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0132] Sulfonyl: -SO2Ra, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfonyl groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0133] Sulfonamide: -SO2NRbRcor -N(Rb)SO2Rc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfonamide groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0134] Sulfonate: -SO3-, wherein the negative charge of the sulfonate group may be balanced with an M+counter ion, wherein M+may be an alkali ion, such as K+, Na+, Li+; anammonium ion, such as+N(Rb)4where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]o.5, [Mg2+]0.5, or [Ba2+]0.5.

[0135] Surfactant: A compound that lowers surface tension between two fluids (liquids and / or gases) and / or between a solid and a fluid. Surfactants can exhibit properties the allow their use as surfactants, dispersants, emulsifiers, foaming agents, wetting agents, lubricants, or a combination thereof.

[0136] Terminating Group: A functional group that is used to terminate an R1group of the formulas according to the present disclosure.

[0137] Thial: -C(S)H.

[0138] Thiocarboxylic acid: -C(O)SH, or -C(S)OH.

[0139] Thiocyanate: -S-CN or -N=C=S.

[0140] Thioester: -C(O)SRaor -C(S)ORawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Thioester groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0141] Thioether: -S-aliphatic or -S-aromatic, such as -S-alkyl, -S-alkenyl, -S-alkynyl, -S-aryl, or -S-heteroaryl; or -aliphatic-S-aliphatic, -aliphatic-S-aromatic, -aromatic-S-aliphatic, or -aromatic-S-aromatic. Thioether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0142] Thioketone: -C(S)Rawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Thioketone groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0143] Triton™ X-100: A compound having a structureIntroduction

[0144] Surfactants play an important role in life science technology and other various industries. With respect to their roles in the life sciences, they are often important components used in both nucleic acid purification and direct PCR processes. For example, in direct PCR, surfactants are needed to enhance the efficiency of the reaction by improving the accessibility of the DNA template. They also can be used to aid in cell lysis, viral inactivation, enhancing DNA accessibility, reducing non-specific binding, and ultimately contributing to the success of obtaining pure nucleic acids and amplifying specific DNA targets. Other processes and / or applications that rely on surfactants are discussed herein and can include, but are not limited to, DNA hybridization, kits for diagnosing allergy-, asthma-, and autoimmune-related diseases, nucleic acid-based sample preparation workflows, and the like.

[0145] Current non-ionic surfactants, such as Triton™ X-100, Tergitol™ NP-40, and / or EH9, that are available as surfactants for myriad biological and / or other industrial uses have a trade-off associated with good performance: they produce degradation by-products that exhibit endocrine disruption effects that interfere with the hormonal system of numerous organisms, particularly aquatic species. These surfactants are often used in sample preparation products, including protein extraction buffers, wash buffers, protein interaction kits, and protein purification kits. To date, no alternative surfactants are available as direct replacements for Triton™ X-100 and / or Tergitol™ NP-40, particularly those that are suitable for life science applications.

[0146] While less hazardous surfactants are available, they do not meet quality or performance standards across a broad range of products or applications, including sensitive biological assays typically used in life science technologies.

[0147] The present disclosure is directed to new compounds that can be used to replace current surfactants, like Triton™ X-100 and / or Tergitol™ NP-40. The disclosed compounds are less hazardous than Triton™ X-100 and / or Tergitol™ NP-40, but do not sacrifice performance as they work as effectively as, or better than, Triton™ X-100 and / or Tergitol™ NP-40. The disclosed compounds can be prepared using cost-effective methods and are biodegradable without producing toxic by-products like the endocrine disrupting by-products that are produced from degradation of Triton™ X-100 and / or Tergitol™ NP-40. The disclosed compounds are suitable for use as surfactants in myriad applications / industries.Compounds

[0148] Disclosed herein are aspects of a compound that exhibits surfactant properties. In some aspects, the compounds of the present disclosure can have a structure according to Formula I.Formula I

[0149] With reference to Formula I, R1is selected to be predominantly hydrophilic, such as comprising a heteroaliphatic group or a combination of a heteroaliphatic group and an aliphatic group; R2and each R4independently is a lipophilic group or a hydrophilic group, provided that if one of R2or (one or more) R4is a hydrophilic group, then R1and the other of R2or (either) R4is selected to provide the compound with a hydrophilic-lipophilic balance (“HLB”) (calculated using Griffin’s Method) value ranging from 10 to 18 (or, if both R2and one or more R4are hydrophilic groups, then R1is selected to provide the compound with such an HLB value); X is selected from oxygen, sulfur, or N(R5); R3is selected from heteroaliphatic, aliphatic, or aryl; each R4independently is aliphatic; Y is selected from oxygen, sulfur, or N(R5); the linker, when present (as indicated with subscript “1” in Formula I), is aliphatic or has a formula -G(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group (e.g., a N-containing heteroaliphatic group, an O-containing heteroaliphatic group, or an S-containing heteroaliphatic group), and X’ is oxygen, sulfur, or N(R5); n is an integer selected from 0 or 1; m is an integer selected from 0 to 6, such as 0, 1, 2, 3, 4, 5, or 6, provided that if n is 0, then m is 1; and p is an integer selected from 0 to 2, such as 0, 1, or 2. With reference to R5groups disclosed above, each R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

[0150] In any of the aspects in the present disclosure, the hydrophilic and lipophilic groups for R1, R2, and / or (one or both of) R4are selected to provide a hydrophilic-lipophilic balance (“HLB”) value ranging from 10 to 18, such as 10 to 12, or 10 to 13, or 11 to 13, or 11 to 14, or 12 to 14, or 12 to 15, or 13 to 15, or 13 to 16, or 14 to 16, or 11 to 18, or 12 to 18, or 13 to 18, or 14 to 18, or 15 to 18, or 16 to 18, or 17 to 18.

[0151] In some aspects, R1comprises a heteroaliphatic group, such as a linear heteroaliphatic group or a cyclic heteroaliphatic group (e.g., a five-membered or 6-membered heterocyclic group). In some aspects, R1can include an aliphatic group in combination with the heteroaliphatic group. In some aspects, R1is a polyalkylene oxide group (e.g., a polyethylene glycol (PEG), a polypropylene glycol, or a combination thereof), a polyalkylene amine group (e.g., a polyethylene amine (PEI), a polypropylene amine, or a combination thereof), or comprises a cyclic C^sheteroaliphatic group, such as a sugar molecule. In some aspects, R1comprises a combination of (i) a polyalkylene oxide group or a polyalkylene amine group or a cyclic C4 sheteroaliphatic group, and (ii) an aliphatic group. In aspects comprising an R1group that is a polyalkylene oxide or a polyalkylene amine, the polyalkylene oxide / amine can be terminated with a hydrogen atom or a terminating group, wherein the terminating group can be an aliphatic group, an aromatic group, or the like. In particular aspects, R1is a PEG or PEI group having a mass average molecular weight ranging from 500 g / mol to 800 g / mol, such as 525 g / mol to 800 g / mol, or 550 g / mol to 800 g / mol, or 575 g / mol to 800 g / mol, or 600 g / mol to 800 g / mol, or 625 g / mol to 800 g / mol, or 650 g / mol to 800 g / mol, or 675 g / mol to 800 g / mol, or 700 g / mol to 800 g / mol, or 725 g / mol to 800 g / mol, or 750 g / mol to 800 g / mol, or 775 g / mol to 800 g / mol. In particular aspects, the PEG group has a mass average molecular weight ranging from 525 g / mol to 575 g / mol, or 725 g / mol to 775 g / mol. In representative aspects, the PEG group has a mass average molecular weight of 550 g / mol or 750 g / mol. In any such aspects comprising a PEG group, the average molecular weights do not include the weight of any terminating group. In aspects comprising a PEG group, the PEG has a formula of -[CH2CH2O]r-, where r is an integer selected to provide a PEG group having a mass average molecular weight as described above. In aspects comprising a PEI group, the PEI has a formula of -[CH2CH2NH]r-, where r is an integer selected to provide a PEI group having a mass average molecular weight as described above. In aspects where R1comprises a cyclic C^sheteroaliphatic group, the cyclic C^sheteroaliphatic group can be bound to X of Formula I directly through a bond formed between X and a carbon atom of the cyclic C^sheteroaliphatic group; or it can be bound to X of Formula I through an external carbonyl group (i.e., -C(=O)-) of the cyclic C^sheteroaliphatic group. In some such aspects, the cyclic C4-sheteroaliphatic group can be a sugar molecule, such as glucuronic acid, trehalose, glucopyranoside, and other sugars, including combinations of such sugars wherein more than one sugar molecule is present (e.g., two to four sugar molecules). In some aspects, R1comprises 1 to 12 PEG groups, or a combination of 1 to 12 PEG groups and an alkyl group (e.g., C1-5 alkyl).

[0153] In any of the aspects in the present disclosure, each of R2or (one or both of) R4independently can be an aliphatic group, an aryl group, a polyalkylene oxide, or a polyalkylene amine. In some other aspects, such as when R1comprises a combination of a heteroaliphatic group and an aliphatic group, one of R2or (one or both) R4comprises a PEG group. In particular aspects, R2is Ci-25alkyl (e.g., C2-2oalkyl, or C2 isalkyl, or Ci-galkyl), phenyl, or naphthyl. In particular aspects, each R4independently is a Ci-ioalkyl group, such as Ci-galkyl (e.g., methyl or ethyl).

[0154] In some aspects, R3is independently is -O-Ci-ealkyl, -O-Csecycloalkyl,

[0155] In one aspect, SS is a solid-support, such as polystyrene (PS), agarose, functionalized magnetic beads, or the like.

[0157] In some aspects, R3is independently is -O-Ci-salkyl, -O-Cs-ecycloalkyl,

[0158] In any of the aspects in the present disclosure, each of R2or (one or both of) R4independently can be an aliphatic group, an aryl group, a polyalkylene oxide, or a polyalkylene amine. In some other aspects, such as when R1comprises a combination of a heteroaliphatic group and an aliphatic group, one of R2or (one or both) R4comprises a PEG group. In particular aspects, R2is Ci-25alkyl (e.g., C2 aoalkyl, or C2 isalkyl, or Ci-galkyl), phenyl, or naphthyl. In particular aspects, each R4independently is a Ci-ioalkyl group, such as Ci-galkyl (e.g., methyl or ethyl).

[0159] In any of the aspects in the present disclosure, R3is selected from an alkoxy group or an aliphatic group (e.g., an alkyl group, an alkenyl group, or an alkynyl group). In some aspects, p is 0, in which case no R3group is present. In other aspects, p is 1 or 2.

[0160] In any of the aspects in the present disclosure, n is 1 and thus the C=Y group is present. In such aspects, Y typically is oxygen or sulfur. In some other aspects, n is 0 and thus the C=Y group is not present and the group bearing X is directly attached to the aryl ring of Formula I.

[0161] In some aspects, X is oxygen or NH. In particular aspects, X is oxygen.

[0162] In any of the aspects in the present disclosure, the linker is not present and thus X is bound directly to R1. In some other aspects, the linker is present. In such aspects, the linker can be a Ci -asaliphatic group (e.g., Ci -ssalkyl) or has a structure according to the formula -C(=Z)-W-C(=Z)-X’-, wherein each Z independently can be selected from O, S, or NR5; W can be Ci-2salkyl, Ci-25alkenyl, Ci-25alkynyl, ether, thioether, or amine (e.g., NH, Ci-25alkyl-N(H)-Ci.25alkyl); and X’ is O, S, or NR5. In particular aspects, the linker, if present, is -(CH2)q-, -C(=O)(CH2)qC(=O)-O-, or -C(=S)(CH2)qC(=S)-O-, -C(=O)N(H)(CH2)qN(H)C(=O)-O-, wherein each q independently is an integer ranging from 1 to 10.

[0163] In any of the aspects in the present disclosure, m is 1, 2, 3, 4, 5, or 6. In particular aspects, m is 1. In any of the foregoing aspects, p is 0, 1, or 2. In particular aspects, p is 0.

[0164] In some aspects, the compound can have a structure according to any one of Formulas IA, IB, IC, ID, IE, or IF, illustrated below.Ri4OxFormula IA Formula IBFormula IC Formula IDRi4Ox-Linker — R mX-RFormula IE Formula IF

[0165] With respect to any of Formulas IA-IF above, each of R1, R2, R3, R4, Y, the linker group, n, m, and p can be as described above for Formula I.

[0166] In some aspects, the compound has a structure according to Formulas IG, IH, I J, and IK, shown below.Formula IJ

[0167] With reference to Formulas IG, IH, IJ, and IK, the aliphatic group is linear, branched, cyclic, or a combination thereof; and the TG group, if present, is an aliphatic group. In particular aspects, the aliphatic group is a linear G2 soalkyl group, such as a linear C21 salkyl group, or a linear C21 oalkyl group; a branched C22oalkyl group, such as a branched C21 salkyl group, or a branched C21 oalkyl group; a cyclic Cs-soalkyl group, such as a cyclic C31 salkyl group, or a cyclic Cs 1 oalkyl group; or a combination of any such linear, branched, and / or cyclic groups. In particular aspects, the aliphatic group is selected from isopentyl, 2-methylpentyl, heptyl, 2,2-dimethylbutyl, pentyl, octyl, nonyl, and the like. The terminating group (or “TG” group) of Formulas IG or IH can be a Ci-ioalkyl group, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl (with any such groups being linear, branched, cyclic, or any combination thereof). In particular aspects, the TG group is methyl. Also with reference to Formulas IG and IH, r is an integer selected to provide a PEG group having a mass average molecular weight as describedabove for Formula I. In some exemplary aspects, r is an integer selected from 2 to 20, such as 2 to 18, or 2 to 16, or 2 to 14, or 2 to 12, or 2 to 10. In particular aspects, r is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0168] Representative compound examples are provided in Table 1, below, wherein the “m-PEG” notations are used to indicate the mass average molecular weight of the PEG group (e.g., “m-PEG 550” is used to indicate a number of PEG repeats that provide a mass average molecule weight of 550 g / mol).OH.00 o 0x00 oMethods

[0169] Disclosed herein is a method for making compounds according to the present disclosure. In some aspects, the method comprises performing chemical reactions to add the lipophilic and hydrophilic groups to a core compound, such as that shown in Scheme 1. In some aspects, the method may comprise one or more synthesis steps to add the lipophilic group. In some aspects, the method may comprise one or more, typically two or more, synthesis steps to add the hydrophilic group. In some aspects, the lipophilic group is added by exposing a starting material 100 (Scheme 1) to a base and an R2-group-bearing electrophile, such as an ^-group-bearing halide compound. In some such aspects, the R2-group-bearing halide compound can be a halide-bearing aliphatic compound, such as Br-aliphatic, l-aliphatic, or Cl-aliphatic. Starting material 100 is then converted to an R2-bearing intermediate, such as intermediate 102 (Scheme 1). Intermediate 102 can then be converted to a compound according to the disclosure that includes the R1hydrophilic group using further synthesis steps.R4Lipophilic Group Hydrophilic Group Addition Addition100 102 1Q4Scheme 1

[0170] In some aspects, intermediate 102 is converted to a compound 104 using a sequence of synthesis steps wherein the aldehyde of 102 is reduced to a hydroxyl group, which is then converted to a suitable leaving group (e.g., a triflate). In such aspects, an R1group can then be added to displace the leaving group, thus providing a compound according to compound 104, wherein n is 0, m is 1, and wherein the linker is not present. Reagents and conditions used in such aspects are recognizable to those in the art, particularly with the benefit of the present disclosure. For example, in some aspects, a suitable reducing agent (e.g., sodium borohydride) can be used to convert the aldehyde to a hydroxyl group. Then a suitable base (e.g., an aminebase) and a suitable leaving group reagent (e.g., methanesulfonyl chloride) can be used to provide the leaving group-containing compound that is reacted with the R1group.

[0171] In yet other aspects intermediate 102 can be converted to a compound 104 by following a similar procedure described above wherein the aldehyde of 102 is reduced to a hydroxyl group. The hydroxyl-containing compound can then be combined with a suitable base and a suitable halide-containing reagent to add the R1group. For example, in some aspects, the hydroxyl-containing compound can be deprotonated with a suitable base and then allowed to react with an R1-halide reagent, such as a halide-bearing sugar molecule, to provide compound 104, wherein n is 0, m is 1, and the linker is not present. In some such aspects, the halide-bearing sugar molecule can be 2-bromo-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

[0172] In yet other aspects, intermediate 102 can be converted to a compound 104 by following a similar procedure described above wherein the aldehyde of 102 is reduced to a hydroxyl group. The hydroxyl-containing compound can then be combined with a suitable base and a suitable linker (or linker precursor) reagent so as to attach a linker group to the hydroxyl group. In some such aspects, the linker can be an aliphatic halide, an anhydride (e.g., succinic anhydride), or other suitable linker groups. Once added, the linker-containing compound can then be reacted under suitable reaction conditions with a suitable R1-containing group to provide compound 104, wherein n is 0, m is 1, and the linker is present.

[0173] In yet other aspects, the aldehyde of compound 100 can be maintained and a suitable X-[Linker]o or 1R1group can be added to the aldehyde using suitable reaction conditions that would be recognized by those in the art with the benefit of the present disclosure. The resulting intermediate could then be oxidized using suitable reagents and reaction conditions to provide a compound 104 wherein N is 1, m is 0 and wherein the linker is or is not present.

[0174] With respect to solid-support (SS) bound surfactants, appropriately functionalized hydroxy-substituted syringic acid derivatives, carboxylic acid substituted syringic acid or hydroxymethyl-substituted syringic acid derivatives are converted to PEG-linked constructs bearing terminal amine or hydroxyl groups. These pegylated intermediates are subsequently coupled to functionalized bead surfaces (for example, carboxyl- or epoxy-activated agarose or polystyrene beads or the like) to generate bead-immobilized non-ionic surfactant analogs.

[0175] Alternatively, polystyrene beads are modified with polyethylene glycol) linkers bearing terminal hydroxyl groups (PS-PEG-OH). Syringic acid derivatives are subsequentlycoupled to the PEG terminus via ester or carbonate linkages to yield PS-PEG-syringic acid derivatives as solid-support bound surfactants. Polystyrene beads functionalized with carboxylic Oacid groups (' — ' ) or hydroxy methyl groups( ' ) can be used to react with the PEG-alcohols to obtain polystyrene beads with PEG linkers bearing terminal carboxylic acid groups or terminal hydroxy groups.Applications

[0176] The disclosed compounds are useful as surfactants and can be used in many applications where a non-ionic surfactant is suggested or preferred. Applications include, but are not limited to, lysing cells, permeabilizing membranes, inactivating viruses, separating hydrophilic proteins from membranes, reducing surface tension, or decellularizing tissue. Additionally, the disclosed compound may be useful as an excipient or adjuvant in a vaccine, a cleaning agent, a component in buffers particularly biological buffers, a wetting agent, an emulsifier, a surfactant, or as a surface treatment for metals. In some aspects, the disclosed compound can be used for DNA or nucleic acid extraction from blood or pathogens applications, protein expression and purification, ELISA’s, Western blotting and RT PCR applications.V. Applications

[0177] The disclosed compounds are useful as surfactants and can be used in many applications where a nonionic surfactant is suggested or preferred. Applications include, but are not limited to, lysing cells, permeabilizing cellular membranes (e.g., cell membranes and nuclear membranes), inactivating viruses, disrupting viral envelopes, reducing non-specific interactions between biomolecules (e.g., proteins and peptides) while maintaining specific interactions between biomolecules (e.g., analyte binding agent and target analytes), separating hydrophilic proteins from membranes, reducing surface tension, enhancing sample wetting, or decellularizing tissue. Additionally, the disclosed compound may be useful as an excipient or adjuvant in a vaccine, a cleaning agent, a component in buffers particularly biological buffers, a wetting agent, an emulsifier, a surfactant, or as a surface treatment for metals.

[0178] In one aspect, the disclosed compounds are useful in methods comprising cell permeabilization. In some aspects, the method comprises treating the cell with the disclosed compound(s) to facilitate cell permeabilization (e.g., permeabilization of the cytoplasmic membrane, the nuclear membrane, or both)._

[0179] In some aspects, permeabilization buffers containing compounds of this disclosure may be used with a cell selected from the group consisting of a eukaryotic cell, a plant cell, a fungal cell, and a bacterial cell. In some aspects, permeabilization buffers containing compounds of this disclosure may be used with a cell selected from the group consisting of blood cells, immune cells, cultured cells, or primary cells.

[0180] In some aspects, membrane permeabilization with the compounds according to the present disclosure can be reversible or irreversible. In some aspects, the permeabilizing agent can be capable of (i) permeabilizing the cell membranes of the cells; and / or (ii) making a cell membrane permeable to allow entry of reagents such as analyte binding reagents, detectable labels, assay reagents, and the like. In some aspects, the permeabilizing agent can comprise, consist essentially of, or consist of a surfactant according to this disclosure, optionally in combination with one or more solvents (e.g., an aprotic solvent). In some aspects, the method can include the removal of the permeabilizing agent (e.g., removing the surfactant according to this disclosure). Removal of the permeabilizing agent can refill the membrane (e.g., reconstitute membrane integrity).

[0181] In one aspect, the disclosed compounds are useful in methods for virus inactivation, particularly inactivating a virus having a lipid envelope, or a virus that may develop a lipid envelope. In some aspects, a method for inactivating a virus comprises combining the disclosed compound with a liquid comprising the virus.

[0182] In other aspects, the disclosed compounds are useful for DNA or nucleic acid extraction such as from blood or pathogens applications, RT PCR applications and the like. In some embodiments, cell lysates or permeabilized cell extrudates and materials obtained by cell lysis or cell permeabilization using compounds of the disclosure and formulations thereof, protein extraction from cell lysates, protein expression and / or purification.

[0183] In some aspects, the compounds of this disclosure can be used in a permeabilization buffer to permeabilize cells, e.g., cells from a sample. Exemplary cells include, but are not limited to adherent cells (for example, HeLa, HEK293, fibroblasts, epithelial cells, neuronal cultures, stem cells), suspension cells (for example, lymphocytes, PBMCs, K562, Jurkat, CHO), primary cells (for example, immune cells, neurons, hepatocytes, endothelial cells), tissue sections (for example, frozen or paraffin-embedded, after fixation and rehydration), microorganisms (for example, yeast, fungi, and bacteria, including both gramnegative and gram-positive), and plant cells. Compounds of this disclosure may also be used in permeabilization buffers for live cells, including mammalian cell lines and primary immune cells (e..g., for calcium flux dyes, intracellular staining, or metabolic assays). In some aspects,compounds of this disclosure may be used in a permeabilization buffer for use with eukaryotic cells, plant cells, fungal cells, bacterial cells, blood cells, immune cells, cultured cells, primary cells, or any cells described above.

[0184] In some aspects, compounds of this disclosure may be used in permeabilization buffers at concentrations of about 0.001 %-10%, (v / v or w / v) concentration depending on the compound chosen. In some aspects, compounds of this disclosure may be used in permeabilization buffers at concentrations of about 0.01 %-5% (v / v or w / v), or 0.05%-1%, or 0.01 %-0.5% (v / v or w / v), or 0.1%-0.5% (v / v or w / v) including 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% (v / v or w / v), and any percentage between.

[0185] In some aspects, the compounds of this disclosure may be included in a permeabilization buffer which may comprise, consist essentially of, or consist of a compound of this disclosure, and one or more of a salt, a buffering agent, a blocking agent, a fixative quencher, or an enzyme.

[0186] In some aspects, the permeabilization buffer includes a buffering agent selected from the group comprising MOPS (3-(N-morpholino)propanesulfonic acid), citrate buffers (saline-sodium citrate (SSC)), phosphate buffers (phosphate buffered saline (PBS)), tris (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-1,3-diol)-based buffers, TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid)-based buffers, HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid)-based buffers, Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid)-based buffers, tricine (N-[tris(hydroxymethyl)methyl]glycine)-based buffers, TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)-based buffers, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)-based buffers, MES (2-(N-morpholino)ethanesulfonic acid)-based buffers, PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid))-based buffers, Cacodylate (dimethylarsenic acid)-based buffers, or any combination thereof.

[0187] In some aspects, the permeabilization buffer includes one or more salts such as monovalent and / or divalent cation salts. In some aspects, the permeabilization buffer may include one or more salts selected from sodium chloride, lithium chloride, ammonium chloride, potassium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, sodium phosphate, magnesium phosphate, tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride, tetrabutylammonium chloride, guanidinium thiocyanate, sodium thiocyanate, or potassium thiocyanate.

[0188] In some aspects, the permeabilization buffer may include a blocking agent which may be selected from known blocking agents, including but not limited to, an agents selected from protein blocking agents, serum blocking agents, mild surfactants (such as the compounds of this disclosure), polymer blocking agents (including polyvinylpyrrolidone (PVP)), and synthetic blocking agents (including commercially available optimized blends). Examples of blocking agents that may be used include bovine serum albumin (BSA), nonfat dry milk, gelatin (fish skin or porcine), normal goat serum (NGS), normal donkey serum (NDS), yeast tRA, homopolymer DNA, denatured salmon sperm DNA, herring sperm DNA, total human DNA, and COTI DNA.

[0189] In some aspects, the permeabilization buffer may include an enzyme. Enzymes may be included, for example, to assist in cell wall digesting, especially in harder to penetrate cells (for example, yeast, bacteria, and plant cells). Enzymes may assist in weakening or partially removing the cell wall for yeast and fungi cells (exemplary enzymes include zymolyase, lyticase, glucanase, and chitinase); they may help with full permeability in bacterial cells (exemplary enzymes may include lysozyme or mutanolysin); and they may be used to remove cellulose and pectin components of plant cell walls (exemplary enzymes include cellulase, pectinase, macerozyme). In some aspects, an enzyme may be a protease to aid in tissue permeabilization or analyte retrieval (exemplary enzymes include proteinase K, trypsin, pepsin, pronase). In workflows involving separation, isolation or analysis of nucleic acids, RNases or DNases may be used (for example, RNase A or DNase I). Particularly in flow cytometry, enzymes used in permeabilization buffers may include protease cocktails (mild digestion for intracellular flow staining). Enzymes used to dissociate tissue can be present in permeabilization buffers (or in dissociation buffers), including collagenase or the like, or in separate tissue dissociation buffers or reagents. Compounds of this disclosure have been shown to work effectively with a wide variety of enzymes, thus making them ideal candidates for surfactants in permeabilization buffers. In some aspects, permeabilization buffers containing compounds of this disclosure may include an enzyme selected from protease, lysis enzyme, trypsin, proteinase K, pepsin, pronase, Papain, lysozyme, zymolyase, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, glucanase, collagenase, dispase, or any combination thereof.

[0190] In some aspects, where samples are subjected to a fixation step, cell permeabilization buffers can include fixative quenchers, e.g., that react with leftover reactive groups from fixatives, in order to stop further cross-linking, reduce non-specific antibody binding, and / or lower background / autofluorescence during downstream analysis, including imaging or flow cytometry. In some aspects, permeabilization buffers containing compounds of this disclosuremay also include fixative quenchers that are useful in the compositions and methods provided herein, including, but not limited to, small amine-containing molecules such as glycine, ammonium chloride, ethanolamine, lysine, and sodium borohydride, serum, bovine serum albumin, or any combination thereof.

[0191] In some aspects, where samples are subjected to a fixation step, cell permeabilization buffers can include fixatives. In some aspects, permeabilization buffers containing compounds of this disclosure may also include fixatives that are useful in the compositions and methods provided herein selected from the group including, but not limited to, paraformaldehyde (PFA) I formaldehyde, methanol, acetone, a methanol / acetone mixture, glutaraldehyde, ethanol, or any combination thereof.

[0192] In some embodiments, cell lysates or permeabilized cell extrudates and materials obtained by cell lysis (e.g., partial cell lysis) or cell permeabilization using compounds of the disclosure and formulations thereof, can be used for applications such as but not limited to immunoassays, ELISAs, protein gel electrophoresis, protein transfer to membranes or supports, live cell assays (including but not limited to mitochondrial function assays, enzymatic activity (e.g., caspase, protease activity), ion-flux assays, cell signaling assays, Ca2+influx assays, NF-kB nuclear translocation assays, and the like), Western blotting, protein detection, protein quantification by colorimetric assays, such as but not limited to BCA (bicinchoninic acid) assays and the like, immunoassay based single plex and multiples methods. Other applications for cell lysates and cell permeabilized materials obtained using compounds of the disclosure include gene expression assays that use signal amplification and are hybridization-based that incorporate for example, branched DNA (bDNA) technology, for measurement of RNA transcripts, e.g., as described in US Patent No. 9783841, or US 10689687.

[0193] In some aspects, when detecting analytes in a cell within a sample, a fixation buffer may be used on the sample before, after, or simultaneously with the cell permeabilization buffer. In some aspects, the sample may be a tissue sample, a cell culture, or an environmental sample. In some aspects, after contacting with the permeabilization buffer and optionally the fixation buffer, the sample may be incubated with an analyte binding agent for a time interval adequate to allow entry of the analyte binding agent into the cell. In some aspects, the method may comprise contacting the sample with a plurality of analyte binding agents, where each analyte binding agent binds to a different analyte, thus providing the ability to analyze multiple analytes in a single sample. The sample may then be optionally washed and analyzed to detect the analyte binding agent(s). The analysis may be direct detection or indirect detection ofthe analyte binding agent. In some aspects, indirect detection may occur when a secondary binding agent is bound to the primary binding agent, and the secondary binding agent is then detected. In some aspects, indirect detection may occur when the sample is contacted with a reagent that produces a detectable signal upon binding to the analyte binding agent, and the detectable signal is then detected. Indirect detection is widely used in immunoassays, microscopy, ELISA, Western blotting, flow cytometry, and similar methods because it amplifies signal and increases sensitivity. The method described can be performed once or multiple times (such as about 5, 10, 15, 20, 25, 30, 35, 40, or more times), and can be used to detect a multiple analytes (e.g., 2-500 different analytes).

[0194] In one aspect, compounds of this disclosure may be incorporated into cell proliferation assay reagents to improve assay consistency, signal quality, and reagent stability. The surfactants may facilitate uniform solubilization of metabolic or fluorescent indicators, reduce nonspecific adsorption of assay components to vessel surfaces, and provide mild permeabilization to enhance access of dyes or substrates to intracellular targets. In endpoint or luminescent proliferation formats, nonionic surfactants may additionally promote efficient cell lysis and support stable enzymatic signal generation. As a result, the inclusion of nonionic surfactants yields improved sensitivity, reduced background, and enhanced reproducibility across colorimetric, fluorescent, and luminescent cell proliferation assay platforms.

[0195] Techniques for accessing and analyzing analytes within cells and / or tissues for subsequent analysis generally employ cell lysis methods that disrupt the cellular compartments. For example, in some methods, lysis media such as ionic surfactants are used, yielding mixtures of cytoplasmic and nuclear content and preventing resolution of molecular information between the compartments (e.g., due to cross-contamination between cytoplasmic mitochondrial DNA and nuclear DNA). However, in many cases it is desirable to analyze analytes within intact (or generally intact) tissues or cells, e.g., to analyze analytes on a sub-cellular level. As such, alternative lysis methods employ mild, nonionic surfactants to disrupt the cellular membrane while leaving the nuclei intact. In another approach, isolated nuclei may be disrupted with digestion enzymes such as proteases. There is a need for reagents that enable the labeling, detection and resolution of molecular information within different parts of cells.

[0196] Permeabilization buffers may be formulated to permeabilize the cell membrane, the nuclear membrane, or both, or membranes of other intracellular organelles, depending on the target analyte. Permeabilization buffers may be formulated to selectively disrupt the plasma membrane while leaving intracellular organelle membranes largely intact. The permeabilization buffer may be tuned to partially permeabilize cells by creating pores specifically in the plasmamembrane, allowing cytosolic components to diffuse out and macromolecules (e.g., dyes, antibodies, nucleic acids) to enter the cell without fully lysing it. This controlled permeabilization is used to access cytosolic targets, deliver probes, prepare semi-intact cells for biochemical assays, and perform immunostaining or subcellular fractionation.

[0197] Cell fractionation broadly refers to a series of processes for separating cellular components into discrete fractions that can be individually analyzed, processed, or utilized in downstream applications. In typical workflows, cells are first disrupted under controlled conditions to release intracellular contents while maintaining the structural integrity of selected organelles. A crude lysate, containing cytosolic material, membrane fragments, and intact organelles, may then be generated through well-known mechanisms. The lysate is then subjected to one or more separation steps under which distinct cellular components segregate based on differences in size, density, or other physicochemical properties. The resulting fractions may include, for example, nuclei, mitochondria, endoplasmic reticulum membranes, plasma membrane components, or soluble cytoplasmic proteins. Nonionic surfactants may be used in the cell fractionation process to achieve selective permeabilization or solubilization of cellular membranes. Nonionic surfactants may be added at defined concentrations to gently disrupt the plasma membrane while preserving the integrity of intracellular organelles, thereby facilitating the isolation of cytosolic fractions with minimal contamination. In other embodiments, nonionic surfactants may be used to selectively solubilize specific membrane populations, enabling enrichment of organellar membranes or membrane-associated proteins without causing substantial denaturation or loss of functional activity. The controlled use of nonionic surfactants thus provides a tunable means of modulating membrane permeability during fractionation, resulting in improved yield, purity, and reproducibility of cellular subcomponents. In some aspects, compounds of this disclosure can be used as alternative nonionic surfactants in cell fractionation workflows as described above.

[0198] In some aspects, the method comprises treating the cell with compounds of this disclosure to facilitate cell permeabilization. In some aspects, compounds of this disclosure may be used in permeabilization buffers that are formulated to permeabilize the cell membrane, the nuclear membrane, or both, or membranes of other intracellular organelles. In some aspects, compounds of this disclosure are used as cell permeabilizing agents in cell imaging methods, including but not limited to, immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), flow cytometry, cell sorting, and spatialomics analysis. In some aspects, the disclosed compound(s) are used as cell permeabilizing agents in cell-based assays including cell proliferation assays, apoptosis assays, cytotoxicity assays, cell viabilityassays, cell cycle assays and caspase assays. In some aspects, compounds of this disclosure may be used as cell permeabilizing agents in nucleic acid detection workflows, e.g., that can include polymerization and / or amplification steps, or detection of nucleic acids in the absence of polymerization and / or amplification. In some aspects, compounds of this disclosure may be used as cell permeabilizing agents on a sample which is then treated sample with a polymerase and / or transcriptase under conditions to permit nucleic acid polymerization and / or amplification.

[0199] In some aspects, compounds of this disclosure may be used in a cell permeabilization buffer contained in a permeabilization kit that can also include one or more reagents selected from the group consisting of: a fixation buffer, a mountant, a wash buffer, an analyte binding agent, a gel, a matrix, a membrane, or any combination thereof.

[0200] In some cases, cell permeabilization buffers may be combined with a blocking buffer either in a single solution or used sequentially.

[0201] Nonionic surfactants are often included in blocking buffers to reduce or prevent non-specific binding or adsorption of assay reagents and / or analytes to that assay components, as well as to inhibit non-specific interactions between polypeptides while maintaining specific binding between analytes and analyte binding agents. In some aspects, compounds of this disclosure can be incorporated into blocking buffers that may be used in connection with any of the techniques or workflows described in this specification or other techniques or workflows (and associated instruments and reagents) where blocking buffers are commonly employed. Such techniques and workflows include but are not limited to ICC, IHC, flow cytometry, single cell assays, fluidics instruments, particle characterization, blotting, Western blot, immunoassays, pulldown assays, sandwich assays, lateral flow assays, and proteomics. Blocking buffers may also be used in other arrays, including microarrays, in situ hybridization (ISH), including FISH, CISH, Southern blot, Northern blot, Western blot and dot blots n some aspects, compounds of this disclosure may be incorporated into a blocking buffer that also includes one or more of a blocking agent and a buffering agent. In one aspect, the blocking agent is non-fat dry milk, albumin, BSA, casein, or gelatin.

[0202] In some aspects the disclosed blocking buffer may be incorporated into a kit that may also include one or more of a wash buffer, a gel, a transfer membrane, a lateral flow strip, and one or more analyte binding agents.

[0203] In some aspects, a blocking buffer comprising compounds of this disclosure may be used by contacting the blocking buffer to a membrane or matrix in an assay, in order to reduce non-specific binding of an analyte binding agent such as an antibody or antibody fragment to a membrane or a matrix in an assay. In some aspects, the membrane or matrix is bound by orembedded with an analyte, prior to the contacting step. In some aspects, the analyte is a polypeptide or a nucleic acid. In some aspects, the blocking buffer may be contacted to the membrane or matrix in a Western Blot, an ELISA assay, a dot blot, a Northern Blot, a Southern Blot, or a lateral flow assay. In some aspects, the claimed method further includes a step where the analyte-bound or analyte-embedded membrane or analyte bound or analyte-embedded matrix is contacted with an analyte binding agent. In some aspects, the membrane or matrix is a nitrocellulose membrane, a PVDF membrane, or an affinity column matrix. In some aspects, the claimed method further includes a washing step where the analyte-bound or analyte-embedded membrane, or the analyte-bound or analyte-embedded matrix, is washed with a wash buffer.

[0204] Mountants are used in a number of techniques and workflows described herein. Mountants include a base medium (e.g., primary fluid, matrix, or bulk material that forms the structural and functional foundation of the mountant formulation) and often include nonionic surfactants. In some aspects, compounds of this disclosure may be incorporated in a mountant formulation in part to reduce surface tension, control nonspecific adsorption of biomolecules, facilitate uniform distribution or penetration of the mountant, and stabilize fluorescent or chromogenic species, thereby improving the clarity, reproducibility, and performance of imaging or analytical assays. In some aspects, the mountant base medium may also include one or more of an anti-fade or fluorophore stabilizer, a refractive index modifier, a preservative, a hardening agent, a wetting agent, or a clearing agent.

[0205] Polypeptides, including but not limited to enzymes, therapeutic proteins, antibodies, antigen peptide ligands, and other biologically or immunologically active proteins, are frequently stored in liquid formulations, however, maintaining polypeptide stability in aqueous compositions presents significant technical challenges. Liquid environments can promote hydrolytic degradation, aggregation, oxidation, deamidation, or conformational rearrangement, any of which can substantially diminish biological activity. Accordingly, the development of stabilized liquid compositions that preserve polypeptide integrity over extended storage periods remains an important objective for many applications. The functional properties of polypeptides are directly dependent on their secondary, tertiary, and — in multimeric complexes— quaternary structures. Even subtle alterations in folding may disrupt catalytic residues, binding domains, or conformational epitopes, leading to reduced efficacy or loss of function. Misfolding or partial unfolding further increases the likelihood of protein aggregation, precipitation, or, in some case, irreversible denaturation. For these reasons, liquid compositions intended for longterm storage usually need to incorporate components that maintain the native conformation ofdiverse polypeptide species under varying environmental conditions, including fluctuations in pH, ionic strength, freeze-thaw cycles, or mechanical stress.

[0206] Detergents, surfactants, and amphiphilic stabilizers, which terms may be used interchangeably herein, have been shown to provide significant protective effects in polypeptide-containing liquid formulations. Nonionic surfactants, such as polysorbates, poloxamers, and other mild surfactants, can prevent protein adsorption to container surfaces, reduce interfacial stress, and mitigate shear-induced denaturation. Surfactants can also stabilize hydrophobic regions of folded proteins, thereby reducing aggregation and promoting retention of native structure. Surfactants are also commonly used in protein storage buffers to prevent microbial growth. In some aspects, the compounds of the present disclosure may be used as alternatives to detergents, surfactants and amphiphilic stabilizers, e.g., in polypeptide storage buffers, reaction buffers, and Master Mixes.

[0207] In enzyme formulations, surfactants may preserve catalytic efficiency by preventing exposure of hydrophobic catalytic pockets to destabilizing interfaces. For antibodies and other large glycoproteins, surfactants can improve colloidal stability and reduce particulate formation during storage and handling. In some aspects, compounds of this disclosure can be used for stabilizing polypeptides or peptides in liquid compositions. In some aspects, compounds of this disclosure may be used in a composition comprising, consisting essentially of, or consisting of a compound of this disclosure in an amount effective to stabilize a polypeptide, the polypeptide, and a buffering agent, and the polypeptide. In some aspects, the composition may further comprise, consist essentially of, or consist of a compound of this disclosure in an amount effective to stabilize a polypeptide, the polypeptide and one or more of a buffering agent, a reducing agent, a chelating agent, a cryoprotectant, a stabilizer or a carrier. In some aspects, the polypeptide is an enzyme, a growth factor, a cytokine, a matrix protein, an antibody or fragment thereof, or an antigen. In some aspects, the enzyme is a nuclease, a polymerase, a reverse transcriptase, a ligase, a glycosylase, a nuclease inhibitor, an alkaline phosphatase, an isomerase, a transferase, an oxidoreductase, and a lyase. In some aspects, the nuclease inhibitor is a DNAse inhibitor or an RNAse inhibitor, or both. In some aspects, the compound of this disclosure is present in the composition in a concentration of about 0.0001%-10% (v / v), or any of 0.001%, 0.005%, 0.01%, 0.03%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% (v / v), or any concentration between. In some aspects, the polypeptide is protein, modified protein or protein derivative (such as protein covalently or non-covalently associated with nonprotein moieties), enzyme, antibody, recombinant or native peptide, monoclonal antibody, polyclonal antibody, antibody fragment or antibody-derived fragment, nanobody, fusion protein,engineered binding scaffold, engineered miniprotein scaffold, vaccine antigen, diagnostic reporter protein, immunopeptide, a structural or carrier protein, signaling peptide, antimicrobial peptide, cell-penetrating peptide, receptor agonist or antagonist peptide, affinity peptide, epitopetag peptide, or a peptide standard.

[0208] In some aspects, compounds of this disclosure can be used in polypeptide storage buffer. In some aspects, compounds of this disclosure can be used in a polypeptide reaction buffer (e.g., an enzymatic reaction buffer). In some aspects, compounds of this disclosure can be used in ready-to-use polypeptide compositions. In some aspects, the polypeptide storage buffer comprises, consists essentially of, or consists of a buffering agent and a compound of this disclosure. In some aspects, the polypeptide storage buffer comprises, consists essentially of, or consists of the a compound of this disclosure, a buffering agent, and one or more of: a reducing agent, a monovalent cation salt, a divalent cation salt, glycerol, a reducing agent, and a chelating agent. In some aspects, the polypeptide storage buffer comprises, consists essentially of, or consists of a buffering agent, a compound of this disclosure, a reducing agent, a chelating agent and glycerol. In some aspects, the composition comprises, consists essentially of, or consists of at least one polypeptide and a polypeptide storage buffer. In some aspects, the composition comprises, consists essentially of, or consists of a mixture of one or more recombinant proteins ranging from 10 kDa to 200 kDa, a buffering agent, a compound of this disclosure, a chelating agent, a reducing agent, a preservative and glycerol. In some aspects the composition comprises, consists essentially of, or consists of, a mixture of one or more recombinant proteins ranging from 10 kDa to 200 kDa, Tris-HCl, EDTA, a compound of this disclosure, SDS, DTT, NaN3and glycerol.

[0209] Many widely used recombinant DNA techniques rely on the catalytic activity of enzymes such as polymerases, restriction endonucleases, ligases, glycosylases, and other DNA modification enzymes. These reactions often occur under conditions that can compromise enzyme stability, including sub-optimal ionic strength, pH variation, and the presence of reaction by-products or contaminants. Accordingly, stabilizing these enzymes during enzymatic reactions is critical to maintaining consistent performance, improving yield, and enabling longer reaction durations or complex multi-step workflows. One way of stabilizing an enzyme under such conditions is to add a stabilizing agent, such as a surfactant. For example, the activity of Taq DNA polymerase has been stabilized by the addition of nonionic surfactants, such as NP-40 or Tween® 20 (Bachmann, et al. Nuc. Acids Res. 18(5): 1309 (1990)). In some aspects, compounds of this disclosure can be used for stabilizing enzymes in their storage buffers. In some aspects, compounds of this disclosure may be used in polypeptide reaction buffers orready-to-use compositions such as master mixes. In some aspects, the polypeptide reaction buffer is an enzyme reaction buffer. In some embodiments, the enzyme component may comprise one or more of polymerases, ligases, nucleases, phosphatases, oxidoreductases, transferases, hydrolases, glycosylases, methyltransferases, or proteases, and / or their recombinant, native, or engineered forms.

[0210] In some aspects, methods for providing an enzyme, a buffering agent and at least one compound of this disclosure and combining the same to form a mixture under conditions such that the activity of the enzyme is stabilized are provided. In some embodiments, at least one compound of this disclosure is included as a component of the present compositions, to provide for both increased stability and activity of the component enzymes. The compounds of this disclosure may be used to maintain a balanced ionic strength and prevent chelation of cofactors and aggregation or inactivation of proteins. The disclosed compounds may be placed into solution at working concentrations and stored according to the methods disclosed herein.

[0211] The compounds of this disclosure may be used in a variety of applications including, for example, nucleic acid amplification reactions. Nucleic acid amplification methods are well known in the art, for example, Polymerase Chain Reaction (PCR), Rolling Circle Amplification (RCA), Loop Mediated Isothermal Amplification (LAMP), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Ligase Chain Reaction (LCR), Self Sustained Sequence Replication (3SR) or solid phase PCR reactions (SP-PCR) such as Bridge PCR etc. (see, e.g., Fakruddin et al., J. Pharm. Bioallied. Sci. 5(4):245-252 (2013) for an overview of the various amplification techniques). The compounds of this disclosure may be used in restriction I cloning I nucleic acid assembly reactions (e.g. Type Ils assembly, Golden Gate assembly, traditional restriction enzyme+ligase cloning), etc. Other applications where the compounds of this disclosure are suitable for use in polypeptide compositions will be apparent to those skilled in the art.

[0212] In some embodiments, the presence of one or more of the compounds of this disclosure may stabilize a polypeptide in a storage buffer. In some aspects, the presence of one or more of the compounds of this disclosure may stabilize a polypeptide within a reaction mixture, decrease inhibition of a polypeptide within a reaction mixture, reduce the adhesion a polypeptide to a surface (such as a wall of a tube, a channel, a chip, a container, or pipette tip, etc.), reduce non-specific activity of a polypeptide during a reaction and / or increase the reaction efficiency of an enzymatic reaction. As such, reaction mixtures comprising, consisting essentially of, or consisting of at least one polypeptide (e.g. an enzyme), a buffer and at least one of the compounds of this disclosure are provided. In some embodiments, a buffering agentmay comprise MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, MES, Tris-HCI, phosphate and / or citrate. In some embodiments, the pH of the composition is about 5.5-9.8.

[0213] In some aspects, compounds of this disclosure may be used in a polypeptide storage buffer composition. The composition may comprise, consist essentially of, or consist of a buffering agent, a polypeptide and a compound of this disclosure and at least one of a reducing agent, monovalent ion salt, divalent ion salt, a chelator, inert protein, crowding agent, amino acids and cryoprotectant. In some aspects, the compound of this disclosure is present in the storage buffer at a concentration of about 0.0001%-10% (v / v), or any of 0.001%, 0.005%, 0.01%, 0.03%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% (v / v), or any concentration between. In some examples the polypeptide storage buffer composition comprises about 1-500 mM buffering agent (such as Tris-HCI, HEPES-NaOH) and about 0.0001-10% (v / v) compound of this disclosure. The polypeptide storage buffer composition may further comprise at least one of about 0.01-100 mM chelating agent (such as EDTA), about 1-70% cryoprotectant (v / v) (e.g. glycerol, sucrose, trehalose, mannitol), about 0.01-100 mM reducing agent (such as DTT), about 0.01-900 mM monovalent salts (such as KCI, NaCI, (NH4)2SO4, LiCI), about 0.01 -900 mM divalent salts (such as MgCl2, MgSO4, MnCl2) and about 0.01-5 mg / ml inert protein (such as BSA). The polypeptide storage buffer may comprise additional stabilizers or additives. The polypeptide concentration in polypeptide storage buffer composition may be about 0.001 -1000 U / pl. In some aspects, the polypeptide in the polypeptide storage buffers described herein is an enzyme or immunopeptide.

[0214] In some aspects, the polypeptide storage buffer composition may comprise T4 RNA polymerase storage buffer comprising about 20 mM Tris-HCI, about 1 mM DTT, about 50 mM KCI, about 0.1 mM EDTA, about 50% (v / v) glycerol, and about 0.03% (v / v) compound of this disclosure. In some aspects, the polypeptide storage buffer composition may be a T7 RNA polymerase storage buffer comprising, consisting essentially of, or consisting of about 20 mM HEPES-NaOH (pH 7.5), about 50 mM NaCI, about 8 mM DTT, about 0.03 % (v / v) compound of this disclosure and about 50% (v / v) glycerol. In some aspects, the polypeptide storage buffer composition comprises, consists essentially of, or consists of uracil-DNA glycosylase storage buffer comprising about 50 mM Tris-HCI (pH 8.0), about 150 mM NaCI, 5 mM DTT, about 0.1 mg / mL BSA, about 0.03% (v / v) compound of this disclosure and about 50% (v / v) glycerol. In some examples the polypeptide storage buffer composition comprises, consists essentially of, or consists of M-MuLV reverse transcriptase storage buffer comprising about 20 mM Tris-HCI (pH 7.5), about 100 mM NaCI, about 0.1 mM EDTA, about 1 mM DTT, about 0.01% (v / v) compound of this disclosure, about 50% (v / v) glycerol. In some aspects, the polypeptidestorage buffer composition comprises, consists essentially of, or consists of phi29 DNA polymerase storage buffer comprising about 330 mM Tris-acetate (pH 7.9 at 37°C), about 100 mM magnesium acetate, about 660 mM potassium acetate, about 1% (v / v) compound of this disclosure, 10 mM DTT. In some aspects, the polypeptide storage buffer composition is RNase A storage buffer comprising, consisting essentially of, or consisting of about 10 mM HEPES (pH 7.2), about 20 mM NaCI, about 0.1% (v / v) compound of this disclosure, 50% glycerol (v / v). In some aspects, the polypeptide storage buffer composition comprises, consists essentially of, or consists of RNase I storage buffer comprising about 50 mM Tris-HCI (pH 8.0), about 100 mM NaCI, about 0.01% (v / v) compound of this disclosure, about 50% (v / v) glycerol. Depending on the specific polypeptide, the polypeptide concentration added to the storage buffer may be about 0.001-1000 U / pil.

[0215] In some aspects, compounds of this disclosure may be included in a reaction buffer for nucleic acid amplification. Reaction buffers provide optimal physicochemical conditions that support the catalytic activity, stability, and specificity of one or more polypeptides during a reaction, such as an enzymatic reaction. In some aspects, compounds of this disclosure may be used in an enzymatic reaction buffer. Enzymatic reaction buffers include, but are not limited to, restriction reaction buffers, and PGR, RT-PCR / RT-qPCR, RT-PCR, or RT reaction buffers. In one aspect, compounds of this disclosure are included in an enzymatic reaction buffer that further comprises, consists of, or consists essentially of one or more of a buffering agent, a polypeptide and, optionally, one or more of a salt, a reducing agent, a chelating agent, a cryoprectant, a stabilizer or a carrier. In some aspects, the compound of this disclosure is present in the enzymatic reaction buffer in a concentration of about 0.0001%-5% (v / v), or any of 0.01%, 0.005%, 0.01%, 0.03%, 0.05%, 0.1%, 0.5%, 1%, 2%, (v / v), or any concentration between. In some aspects, compounds of this disclosure may be included in a reaction buffer for Polymerase Chain Reaction (PGR) or RT-PCR / RT-qPCR (reverse transcription - polymerase chain reaction, quantitative reverse transcription - polymerase chain reaction). In some aspects, the PGR reaction buffer comprises, consists essentially of, or consists of Tris-HCI, (NH4)2SO4, a compound of this disclosure and a DNA polymerase enzyme. In case of RT-PCR reaction composition, reverse transcriptase enzyme is added to the PGR reaction composition (e.g. in one-step RT-PCR settings). In a two-step RT-PCR settings, the reverse transcription reaction is conducted first and then all or a portion of RT reaction composition is used in the subsequent PCR reaction. An exemplary 5x RT reaction buffer can include: 250 mM Tris-HCI (pH 8.3), 375 mM KCI, 15 mM MgCl2. The RT reaction buffer may further comprise additives,such as salts or surfactants. In some aspects, the surfactant in the RT reaction buffer may be a compound of this disclosure. In some aspects, a suitable 10x PCR reaction buffer may comprise, consist essentially of, or consist of, about 750 mM Tris-HCI, about 200 mM (NH4)2SO4, about 0.1% (v / v) compound of this disclosure. The buffer may further comprise 20 mM MgCl2. In some aspects, a compound of this disclosure is included in a restriction reaction buffer composition. In some aspects, a restriction reaction buffer may comprise, consist essentially of, or consist of about 33 mM Tris-acetate (pH 7.9 at 37°C), about 10 mM magnesium acetate, about 66 mM potassium acetate, about 0.1 mg / ml BSA and about 0.1% (v / v) compound of this disclosure. The skilled artisan will readily appreciate that the compounds of this disclosure are useful in a variety of downstream assays, e.g., that do not require downstream polymerization or amplification reactions. By way of example, the compounds of the present disclosure can be used in branched DNA-based assays to detect target nucleic acids present in a sample lysate, e.g., as described in US Patent No. 8,426,578. Depending on the specific reaction, the enzyme concentration added to the reaction buffer may be about 0.001-1000 U / pl. The buffer formulations described herein are provided by way of example, and the skilled person will understand that the composition may be optimized as necessary, using conventional methods and without departing from the scope of current disclosure.

[0216] In some aspects, compounds of this disclosure may be incorporated in a ready-to-use reaction mixture or a Master Mix and said composition may comprise, consist essentially of, or consist of at least one polypeptide (e.g., an enzyme), a buffering agent, a compound of this disclosure, and optionally monovalent and / or divalent cation salt(s) and nucleotides. In some aspects, the polypeptide incorporated in the Master Mix is an enzyme. In some aspects, a Master Mix may further comprise, consist essentially of, or consist of, one or more of a chelating agent, a reducing agent, a cryoprotectant, an inert protein, a dye, one or more primers. In some aspects, the Master Mix comprises, consists essentially of, or consists of a polymerase enzyme, a buffering agent, magnesium salt, ammonium salt, BSA (bovine serum albumin), a compound of this disclosure, dNTPs. In some aspects, a PCR Master Mix comprises, consists essentially of, or consists of, Taq DNA polymerase, Tris-HCI, MgCI2, KCI, BSA, compound of this disclosure, dNTP.

[0217] In some aspects, compounds of this disclosure may be incorporated in a Master Mix for DNA fragment end conversion in library preparation workflow. In some aspects, the Master Mix comprises, consists essentially of, or consists of, a compound of this disclosure, enzymes, buffers and other necessary reaction / storage components in the following 1X, 2X, 3X, 5X, or 10X concentrations and ratios: about 1 U / pil T4 polynucleotidekinase; about 0.32 U / pl T4 DNA polymerase; about 0.12 U / pil Klenow fragment, and about 0.2 U / pl mod- Tbr DNA polymerase. Reaction co-factors and nucleotides: about 20 mM MgCk 2 mM dATP; about 0.4 mM dCTP; about 0.4 mM dTTP; about 0.2 mM dGTP; about 2 mM ATP. Ionic strength and pH of the End Conversion Master Mix may be formulated using about 100 mM - 105 mM Tris-HCI, pH 8.3 and monovalent metal hydrochloric acid salts, e.g. NaCI, KCI, LiCI, at 20 mM to 50 mM, stabilizers and cryoprotectants: about 20 mM DTT; about 0.2%-0.4% (v / v) compound of this disclosure; about 12% (v / v) glycerol; and about 0.024 mM EDTA.

[0218] Other salts and stabilizers suitable for use in polypeptide compositions will be apparent to those skilled in the art and may differ for different polypeptides used in selected reactions. In some aspects, the polypeptide stabilizing compositions may be supplemented with additional stabilizers and / or cryoprotectants. In some aspects, the composition is further lyophilized or air-dried. Air drying involves drying under conditions of ambient or elevated temperatures at atmospheric pressure. Lyophilization is a drying process in which water molecules are removed from a frozen solution under a vacuum. Typical stabilizers suitable for use in lyophilization or air drying include disaccharides such as trehalose and sucrose, polysaccharides such as dextran or pullulan, polyols such as mannitol, sorbitol, amino acids including glycine and arginine, as well as proteins such as bovine serum albumin (BSA), human serum albumin (HSA) or gelatin. Additional stabilizing agents may comprise polyethylene glycol (PEG) and / or surfactants (such as the compounds of this disclosure). The choice and concentration of these excipients can be adjusted to optimize polypeptide recovery and activity after lyophilization or air drying. A person skilled in the art will readily understand how to modify the formulation and drying conditions to ensure compatibility of the specific polypeptide system with either lyophilization or air-drying processes. In some aspects, the polypeptide disclosed herein is an enzyme. A composition prepared for lyophilization or airdrying may comprise at least one of trehalose, sucrose, maltitol, PEG, mannitol, dextran, Ficoll™, bovine serum albumin (BSA) and / or human serum albumin (HSA).

[0219] In some aspects, compounds of this disclosure may be present in storage buffers for resins, such as chromatography resins, particles, or beads. In some aspects, compounds of this disclosure may be present in storage buffers for resins, such as ch omatography resins, particles, or beads wherein a polypeptide is attached to the resin or bead. In some cases, a bead or microcarrier may be conjugated to an analyte binding agent for use in analytical assays requiring selective capture, isolation, or detection of one or more target analytes. The conjugated beads or microcarriers enable high-affinity binding interactions and may be incorporated into heterogeneous assay formats, including, for example, flow cytometric analysis,magnetic separation, immunoassays, high-throughput or multiplexed analytical systems, and nucleic acid detection workflows. In certain embodiments, the conjugated beads or microcarriers facilitate enhanced signal-to-noise ratios by concentrating the analyte on a solid support, thereby improving analytical sensitivity, specificity, and throughput. These conjugated beads or microcarriers are kept in solutions that often include nonionic surfactants to enhance the physical and functional stability of the conjugates; reduce nonspecific adsorption of proteins and other biomolecules to the bead or microcarrier surface and to container interfaces, thereby preserving the active binding sites of the conjugated agent; minimize aggregation of beads or microcarriers by lowering interparticle surface tension and providing a steric barrier that prevents bead-bead or microcarrier-microcarrier interactions during storage, handling, and assay implementation; and / or maintain the dispersion state of the beads or microcarriers under thermal, mechanical, or freeze-thaw stress, resulting in improved assay reproducibility and extended shelf life of the conjugated bead or microcarrier composition. The compositions of this disclosure may be incorporated into a storage buffer for a number of beads or microcarriers, particularly ones that are conjugated to an analyte binding agent. Accordingly, provided herein are compositions that comprise, consist essentially of, or consist of beads or microcarriers (e.g., that are coupled to or conjugated to analyte binding agent), and a surfactant of the present disclosure. The disclosed compositions and methods are compatible with automated liquid-handling platforms and diverse detection modalities, permitting integration into high-throughput or multiplexed analytical systems. In some aspects, compounds of this disclosure may include a composition containing compounds of this disclosure and a bead or microcarrier conjugated to an analyte binding agent. In some aspects, compounds of this disclosure may include a composition comprising, consisting essentially of or consisting of compounds of this disclosure and multiple beads or microcarriers conjugated to two or more analyte binding agents that selectively target two or more analytes. In some aspects, the analyte binding agent is a capture antibody reagent. In some aspects, the compounds of this disclosure are in a composition that may also comprise, consist essentially of, or consist of one or more of a buffering agent, a reducing agent, a monovalent cation salt, a divalent cation salt, glycerol, a reducing agent, and a chelating agent. In some aspects, the composition may include a compound of this disclosure in a concentration of about 0.0001 %-10% (v / v), or any of 0.001%, 0.005%, 0.01%, 0.03%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% (v / v). In some aspects, compounds of this disclosure may may be incorporated in a kit that includes a composition containing compounds of this disclosure and a bead or microcarrier conjugated to an analyte binding agent.

[0220] In some aspects, the bead or microcarrier may be selected from a group including, but not limited to, magnetic beads (streptavidin, Ni-NTA / His-tag, carboxylated, Protein A / G, ionexchange, silica-coated), silica beads, agarose resins or beads including Sepharose (Protein A / G agarose, glutathione agarose, Ni-NTA agarose, activated agarose), polymer beads (ionexchange, hydrophobic interaction, reversed-phase / C18), paramagnetic nanoparticles, specialty beads such as immunomagnetic beads, lectin beads, aptamer-coated beads, and oligo(dT) beads, hydroxyapatite beads, cellulose and modified cellulose resins, cellulose beads, dextran-based (Sephadex) beads, polyacrylamide beads, chitosan-coated beads, modified polysaccharide beads, polystyrene-divinylbenzene (XAD) beads, ceramic or zirconia beads, alumina beads, enzyme-immobilized beads, metal oxide beads (Fe3O4, TiO2, ZrO2, AI2O3), sialic-acid-binding beads, graphene oxide or carbon-based beads, MOF-based beads, polystyrene beads and resins, and many more. Examples of non-bead particles used for binding target molecules include nanoparticles (gold, silver, silica, iron oxide), quantum dots, liposomes, polymeric nanoparticles, carbon nanotubes, graphene or graphene oxide sheets, metal-organic frameworks (MOFs), micelles, dendrimers, magnetic nanorods or nanowires, hydrogels or nanogels, porous silica particles, and aptamer-coated nanoparticles. In some aspects, the solid support is selected from PEG-based beads such as TentaGel and pegylated-polystyrene beads, silica and silica-gel supports including functionalized silica beads, alumina supports, magnetic silica or polymer-coated magnetic beads, controlled-pore glass beads, functionalized magnetic beads, functionalized silica gel, chitosan beads, and pre-functionalized systems including NHS-activated beads, maleimide-activated beads, epoxy-activated supports, and streptavidin-biotin capture systems, maleimide-activated beads, and epoxy-activated supports.

[0221] In some aspects, compounds of this disclosure may be incorporated in a kit comprising, consisting essentially of, or consisting of (1) a capture antibody reagent, wherein the capture antibody reagent comprises a bead or microcarrier conjugated to a first analyte binding agent, wherein the analyte binding agent is an antibody or fragment thereof or a non-antibody capture agent that specifically binds a first epitope on a target analyte; (2) a detection antibody reagent, wherein the detection antibody reagent comprises a detectable label, wherein the second analyte binding agent specifically binds a second epitope on the target analyte; and optionally a lysis buffer; optionally a wash buffer; wherein one or more of the capture antibody reagent, the detection antibody reagent, the lysis buffer, or the wash buffer comprises a compound of this disclosure. In some aspects, the capture antibody reagent comprises a bead or microcarrier that is internally labeled with a dye.

[0222] In some aspects, a compound of this disclosure may be used as a stabilizing agent in the cell culture in recombinant protein manufacturing processes. Secreted recombinant proteins may for example aggregate and / or misfold after secretion I cell lysis. To stabilize a protein's structure, a compound of this disclosure may be added as a stabilizing agent before, during or after fermentation. In some aspects, a protein affinity chromatography purification running and / or elution buffer may comprise a compound of this disclosure. An exemplary elution buffer may comprise, consist essentially of, or consist of 10-100 mM NaFhPC, 100-500 mM NaCI, 100-500 mM imidazol, pH 8.0, 1-100 mM p-mercaptoethanol and 0.01-1% (v / v) compound of this disclosure.

[0223] In some aspects, compounds of this disclosure may be used in polypeptide purification workflows and associated reagents. For example, compounds of this disclosure can be included in sample, equilibration, wash, elution or storage buffers at concentrations comparable to those used for Triton™ X-100 and related surfactants, but without the regulatory and environmental disadvantages associated with alkylphenol ethoxylates.

[0224] In some aspects, the compounds of this disclosure can be used in a broad range of chromatographic protein purification workflows. Such workflows may include ion exchange_chromatography, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, reverse phase chromatography, immunoaffinity chromatography, or mixed mode chromatography. In some aspects the compounds of this disclosure are suitable for use in immobilized metal affinity chromatography (IMAC), ion exchange chromatography using strong anion exchangers such as Q-type resins, and other column formats where non-ionic surfactants are commonly included to maintain solubility and prevent non-specific binding. Exemplary working concentrations of nonionic surfactants such as Triton™ X-100 in biochemical and protein purification buffers are in the range of about 0.01-2% (w / v or v / v), for example 0.1-1% in lysis buffers, with lower concentrations (e.g. 0.01-0.1%) often employed in binding, wash, and storage buffers. In some aspects, compounds of this disclosure may be used at analogous levels, for example at about 0.01-2% (w / v), about 0.05-0.5%, and about 0.1 -0.2% (w / v), depending on protein class and chromatography step.

[0225] In some aspects, the compounds are used in IMAG workflows for the purification of native or recombinant polypeptides, including His-tagged recombinant proteins and naturally histidine-rich proteins. Exemplary IMAC resins suitable for use include, without limitation: Ni Sepharose™ High Performance and Ni Sepharose™ 6 Fast Flow (Cytiva), which are agarose-based nickel-charged resins with high binding capacity for histidine-tagged proteins and broadcompatibility with common buffer additives, including surfactants, denaturants (e.g. urea, guanidinium-HCI), and reducing agents; Profinity™ IMAC resins (Bio-Rad), available as Ni2+-charged iminodiacetic acid (IDA) resins that are compatible with a wide range of salts, surfactants and denaturants, including 8 M urea and 6 M guanidinium-HCI; Ni-NTA Agarose and Ni-NTA Superflow resins (QIAGEN), nickel-charged nitrilotriacetic acid (NTA) resins for purification of 6xHis-tagged proteins under native or denaturing conditions; and other commercial Ni2+resins, such as HisPur™ Ni-NTA (Thermo Fisher Scientific) or Ni-NTA agarose resins from additional suppliers. Compatibility tables for such resins show tolerance to nonionic surfactants at concentrations up to about 1-2% (w / v). Compounds of this disclosure do not negatively affect resin appearance at concentrations typical for protein purification, binding capacity or backpressure, and do not interfere with monovalent salts, urea, guanidinium-HCI or other buffer components commonly used in chromatography workflows.

[0226] In other embodiments, compounds of this disclosure are used in ion-exchange chromatography, particularly with strong anion exchange (Q-type) resins. Exemplary resins include: Q Sepharose™ Fast Flow (Cytiva), a strong anion exchanger based on a 6% cross linked agarose matrix functionalized with quaternary ammonium (Q) groups, widely used for capture and intermediate purification of proteins at laboratory and industrial scale; related ion exchangers such as SP Sepharose™ Fast Flow (strong cation exchanger), DEAE Sepharose™ Fast Flow (weak anion exchanger), and CM Sepharose™ Fast Flow (weak cation exchanger), which share a robust agarose Fast Flow matrix and are similarly compatible with typical chromatography additives. Compounds of this disclosure may be used (e.g. in concentrations of 0.01-0.5% w / v) in Q- or SP-type ion-exchange buffers, to maintain column performance and protein recovery while providing improved protein solubility and reduced non-specific adsorption, without detrimental interaction with Q-type resin ligands, resin matrices, or common buffer components (such as NaCI, phosphate, Tris, urea or guanidinium-HCI).

[0227] In some aspects, compounds of this disclosure may be suitable for use in other chromatographic techniques where gentle surfactants are advantageous. For example, compounds of this disclosure may be used in size exclusion chromatography on agarose- or dextran-based resins to prevent aggregation and non-specific interactions; hydrophobic interaction chromatography, where low concentrations of nonionic surfactants can suppress nonspecific hydrophobic sticking while preserving desired binding or mixed-mode and affinity resins, including immunoaffinity columns where maintenance of conformational epitopes is critical for downstream immunoassays.

[0228] In some aspects, compounds of this disclosure may be used in chromatography running buffers and chromatography elution buffers, including for chromatographic techniques discussed here, as well as others that are known to one of skill the art. In some aspects, compounds of this disclosure may be used in chromatography running buffers and chromatography elution buffers that are used with various chromatography columns and matrixes, including those comprising resins described herein and those known in the art. In certain embodiments, the elution buffer may also comprise salts, pH-adjusting agents, organic solvents, surfactants, chelators, reducing agents, stabilizers, or other functional components that promote selective recovery of bound molecules. The elution buffer may be applied in a stepwise, gradient, or continuous format and is suitable for use in affinity, ion-exchange, hydrophobic interaction, mixed-mode, reverse-phase, or other chromatographic systems. In one aspect, the disclosed chromatography elution buffers and chromatography running buffers may further comprise one or more of a buffering agent, a polypeptide and, optionally, one or more of a salt, a reducing agent, a chelating agent, a cryoprectant, a stabilizer or a carrier. In some aspects, the compound of this disclosure is present in the composition in a concentration of about 0.005-5% w / v, 0.05%-1%, or any concentration between.

[0229] In some aspects, a compound of this disclosure is used as a surfactant that facilitates the formation and stabilization of water-in-oil emulsions suitable for use, e.g. in in vitro compartmentalization (I VC) and compartmentalized self-replication (CSR) processes and its modifications (see, e.g., US9,683,251 which is incorporated herein by reference). The surfactant acts at the aqueous-organic interface to reduce interfacial tension and to generate discrete, stable aqueous microdroplets dispersed within a continuous oil phase. Each microdroplet thereby defines a microcompartment capable of maintaining biochemical reactions in isolation from other compartments. In some examples, the aqueous phase comprises nucleic acids, enzymes, ribosomes, and / or reaction substrates, such that the surfactant-stabilized droplets serve as self-contained microreactors wherein a nucleic acid and its encoded product remain physically associated. This configuration enables genotype-phenotype linkage during selection or amplification steps. Accordingly, the surfactant operates as a critical structural element in maintaining compartmental integrity and reproducibility of the emulsion-based workflow. In some aspects, an exemplary CSR mix for DNA polymerase mutagenesis may comprise, consist essentially of, or consist of a reaction buffer, primers, dNTPs, induced E.coli cells overexpressing mutant polymerases (DNA polymerase gene can be mutated using error prone PCR). The mutated gene library can be transformed into selected Escherichia coli cells added to oil phase containing 2% (v / v) ABIL EM 90, 0.01-0.1 % (v / v) compound of this disclosure in mineraloil under constant stirring (1700 rpm) at +4° C. After addition of the aqueous phase (gradually over two min), stirring may be continued for five min. The emulsion may then be frozen at -80° C. and thawed at 37° C-50° C (temperature increased gradually after each selection round). Several freezing-thawing cycles can be performed. The emulsion then can be incubated for 16-2 hours depending on the conditions of the selection rounds.

[0230] Immunohistochemistry (IHC) is a tissue-based technique that uses analytespecific analyte binding agents, such as antigen-specific antibodies, to detect and visualize proteins in situ within form al in -fixed, paraffin-embedded or frozen sections, preserving tissue architecture while providing spatial information on protein expression and localization. Throughout IHC workflows, nonionic surfactants are used to control tissue permeability, analyte binding agent, e.g., antibody, access, and nonspecific interactions. Mild nonionic surfactants are commonly included in wash and blocking buffers to reduce hydrophobic binding of analyte binding agents, e.g., antibodies, to tissue and glass, lower background staining, and improve reagent penetration through the section. For some analytes — especially intracellular or membrane-associated epitopes — stronger or more permeabilizing nonionic surfactants such as Triton™ X-100 (or newer substitutes) or low levels of SDS may be used after fixation and analyte / antigen retrieval to partially disrupt lipid membranes and enhance analyte binding agent diffusion into dense tissue regions. Nonionic surfactants are also incorporated in analyte retrieval and decloaking solutions or in automated Stainer reagents to standardize wetting and rinsing. As in other immunoassays, their type and concentration must be carefully optimized: insufficient surfactant can lead to poor penetration and high background, while overly aggressive surfactancy can extract membrane proteins, distort morphology, or destroy sensitive epitopes, compromising both signal intensity and histological interpretation.

[0231] In some aspects, compounds of this disclosure may be incorporated in these IHC workflows and associated reagents, including in wash buffers, in blocking buffers, in mountants, and the like, used to disrupt lipid membranes and enhance antibody diffusion after fixation and antigen retrieval, or incorporated into analyte retrieval solutions, decloaking solutions, or automated Stainer reagents. In some aspects, compounds of this disclosure may be included in analyte binding reagent dilution buffers, e.g., antibody dilution buffers used with immunoassays, including IHC, to prevent analyte binding agents (e.g., antibodies) sticking to walls, glass, or plastic; to help even distribution over the tissue section; and to prevent non-specific binding as well as improve the overall sensitivity of the assay.

[0232] Spatial biology is a rapidly advancing field focused on characterizing biomolecules within intact tissues while maintaining their native spatial relationships. Techniques such as immunohistochemistry (IHC), immunofluorescence (IF), and multiplexed imaging are widely used to resolve cellular organization, tissue architecture, and molecular interactions, and are foundational in immuno-oncology, pathology, neuroscience, and developmental biology. The performance of these assays, however, can be limited by endogenous tissue autofluorescence, autofluorescence of certain surfactants (e.g., Triton™ X-100 and the like), and by insufficient permeabilization or surfactant-mediated processing, which collectively reduce multiplex assay sensitivity, specificity, and overall reliability. For example, spleentissue exhibits high autofluorescence and dense lymphoid architecture with abundant macrophages, leading to nonspecific binding, elevated background (especially when used with surfactants having inherent autofluoresence), and uneven reagent penetration that impair staining consistency and complicate interpretation in multiplex workflows.

[0233] Nonionic surfactants are commonly used in IHC and spatial biology to enhance tissue permeabilization while preserving morphology and antigenicity. They can improve penetration of antibodies and detection reagents, reduce nonspecific binding and autofluorescent background, and promote uniform staining across structurally complex tissues. In multiplex and cyclic IHC formats, nonionic surfactants also facilitate efficient reagent exchange and maintain tissue integrity over repeated processing steps, thereby improving signal clarity and reproducibility in high-plex assays. Compounds of this disclosure can be applied in multiplex IHC workflows across multiple tissue types, including skin and spleen, and in someinstances exhibit comparable or superior signal and staining performance relative to Triton™ X-100. These improvements support the use of the disclosed surfactants in high-complexity spatial biology applications where signal discrimination and panel robustness are critical.

[0234] Immunocytochemistry (ICC) is a microscopy-based technique that uses analytespecific analyte binding agents (e.g., antigen-specific antibodies) to detect and localize proteins within fixed cells, providing spatial information on protein expression, trafficking, and cell state at single-cell resolution. A critical component of many ICC protocols is the inclusion of surfactants — amphiphilic molecules that modulate membrane permeability, protein-surface interactions, and solution properties. Nonionic surfactants such as Tween20 and Triton™ X-100 are widely used during permeabilization and wash steps to gently disrupt lipid bilayers and enhance antibodypenetration while reducing nonspecific hydrophobic interactions that contribute to background staining. More selective agents like saponin and digitonin interact primarily with cholesterol-rich membranes, enabling reversible permeabilization of the plasma membrane while better preserving intracellular organelle integrity, whereas stronger ionic surfactants such as SDS are generally avoided or used at very low concentrations due to their denaturing effects on proteins and epitopes. Nonionic surfactants can facilitate the entry of analyte binding agents such as antibodies or the like into intracellular compartments while preserving cellular architecture and antigenicity. In addition to permeabilization, low concentrations of nonionic surfactants in blocking and wash buffers help prevent analyte binding agent (e.g., antibody) aggregation and adsorption to plastic or glass surfaces, thereby improving signal-to-noise ratios. There exists a need for nonionic surfactants with reduced toxicity and improved environmental profile compared to, for example Triton™ X-100, that are compatible with ICC workflows. In some aspects, compounds of this disclosure may be used selectively throughout ICC workflows, including in cell permeabilization, in blocking buffers, in wash buffers, in mountants, and generally stabilizing analyte binding agent (e.g., antibody) diluent formulations and other protein reagents during ICC workflow.

[0235] Multiplex ICC enables simultaneous visualization of multiple biomarkers and cellular structures, providing a comprehensive view of cell architecture, function, and pathology that supports diagnostic evaluation, therapeutic stratification, and high-content drug screening.However, achieving reliable multiplex detection is technically challenging because differing fixation, permeabilization, and antigen-preservation requirements can disrupt cellular structures, limit epitope accessibility, interfere with protein-protein interactions, and produce unbalanced or inconsistent signal across targets. For example, when detecting targets on both the cell surface and intracellular targets, milder surfactants may not penetrate into some intracellular areas, while harsher surfactants can cause conformational changes in the surface target proteins and lead to an altered staining pattern.

[0236] For example, concurrent detection of Mitofilin and ZO-1 offers valuable insight into the coordinated disruption of mitochondrial integrity and tight-junction organization characteristic of many diseases, yet it requires carefully balanced conditions to preserve both mitochondrial and junctional morphology while enabling accurate assessment of disease mechanisms and therapeutic responses. These difficulties are further compounded by the limitations of conventional nonionic surfactants, which either insufficiently permeabilize inner mitochondrial membranes or, when used at higher concentrations, disrupt tight-junction architecture andcytoskeletal organization. Furthermore, conventional surfactants may autofluoresce and interfere with analyses utilizing certain detectable labels.

[0237] Difficulties increase further when cytoskeletal and nuclear stains are incorporated, as the combined sensitivity of mitochondrial, junctional, cytoskeletal, and nuclear components to different surfactants and fixation chemistries sharply narrows workable staining conditions. Multiplex ICC workflows spanning these compartments must overcome substantial technical constraints — amplified by the limitations of known surfactants — to achieve robust, interpretable, and accurate co-localization. Compounds of this disclosure have demonstrated effectiveness when used for multiplex ICC, and in some cases have demonstrated equivalent or better performance as compared to leading commercial surfactants. Advantageously, some disclosed compounds are effective at significantly lower concentrations, reducing surfactant by-product and offering further workflow advantages over other alternatives.

[0238] Flow cytometry is a high-throughput, single-cell analytical technique that measures light scatter and fluorescence as cells flow past a focused laser beam, enabling detailed phenotyping of heterogeneous cell populations. Nonionic surfactants play several important but carefully controlled roles in flow cytometry workflows. Nonionic surfactants such as Tween 20 and Triton™ X-100 are commonly used in intracellular staining buffers to permeabilize cell membranes just enough to allow analyte binding agent (e.g., antibody) access to cytoplasmic or nuclear targets while preserving overall cell structure and epitope integrity. In staining and wash buffers, low levels of nonionic surfactants help minimize nonspecific binding and reduce cell aggregation by limiting hydrophobic interactions and cell-plastic adhesion, which improves data quality and prevents clogging of the flow cell. Some formulations for lysing red blood cells or fixing / permeabilizing leukocytes combine fixatives with nonionic surfactants to streamline sample preparation for whole-blood immunophenotyping. There exists a need for more eco-friendly nonionic surfactants for use in flow cytometry workflows that do not compromise membrane integrity, alter light-scatter properties, reduce viability in livecell assays, or denature sensitive antigens, yet still provide for strong and consistent intracellular staining. In some aspects, compounds of this disclosure may be used throughout the flow cytometry workflows, e.g., in associated reagents, as described above.

[0239] Multicolor flow cytometry is an analytical technique that enables simultaneous measurement of multiple cellular features — such as phenotype, activation state, and functional markers — by labeling cells with distinct fluorophore-conjugated antibodies. Multicolor flowcytometry experiments often require the simultaneous detection of multiple cell-surface markers (e.g., CD antigens) together with intracellular or intranuclear targets. Conventional permeabilization methods frequently compromise surface-epitope integrity, while surface-staining protocols may restrict antibody access to intracellular components, creating a need for reagents that can reconcile these competing requirements. Certain nonionic surfactants may aid in this process, due to their ability to gently disrupt lipid membranes without inducing significant protein denaturation or epitope masking. Compounds of this disclosure are effective surfactants for such techniques, demonstrating an ability to transiently permeabilize the plasma and intracellular membranes while preserving the conformational stability of surface antigens and maintaining overall cell morphology, while advantageously not leading to increased background, as with some conventional surfactants such as Triton™ X-100.

[0240] In some embodiments, provided herein is a method for analyzing cells in a sample via flow cytometry that includes the steps of: fixing (e.g., with PFA, formalin) and permeabilizing cell membranes (e.g., with a compound according to the present disclosure) prior to contacting the cells with a plurality of analyte binding reagents. Fixation can be performed using a fixating agent, such as a cross-linking agent. The fixing agent can comprise a cleavable cross-linking agent, e.g. that includes a thiol-cleavable cross-linking agent. The cleavable crosslinking agent can comprise or be derived from dithiobis(succinimidyl propionate) (DSP, Lomant's Reagent), disuccinimidyl tartrate (DST), Bis [2-(Succinimidooxycarbonyloxy)ethyl] Sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), dimethyl 3,3'-dithiobispropionimidate (DTBP, Wang and Richard's Reagent), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-a(2-pyridyldithio)toluene (SMPT), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), succinimidyl 2-((4,4'-azipentanamido)ethyl)-1,3'-dithiopropionate (SDAD, NHS — SS-Diazirine), or any combination thereof. The cleavable crosslinking agent can comprise a cleavable linkage selected from a chemically cleavable linkage, a photocleavable linkage, an acid labile linker, a heat sensitive linkage, an enzymatically cleavable linkage, or any combination thereof. The cleavable cross-linking agent can comprise a disulfide linker. The fixing agent can comprise a non-cross-linking fixative, optionally the non-cross-linking fixative comprises methanol. The method can comprise contacting the cells with an unfixing agent. The unfixing agent can comprise a thiol, hydoxylamine, periodate, a base, or any combination thereof. The unfixating agent can comprise DTT. The unfixing agent can, in some embodiments, cleave a disulfide bridge. The unfixing agent can reverse the fixation during a lysis step. After treatment with the analyte binding agents, the permeabilized cells optionally may besubjected to flow. This process may be used in a number of workflows, such as ICC, IHC and flow cytometry.

[0241] Single-cell assays are analytical methods configured to resolve biological, molecular, or functional characteristics at the level of individual cells, including but not limited to single-cell RNA sequencing (scRNA-seq), single-nucleus RNA sequencing (snRNA-seq), singlecell DNA sequencing, single-cell exome or genome sequencing, single-cell ATAC-seq, single-cell chromatin immunoprecipitation assays (scChlP-seq), single-cell methylation and other epigenomic assays, single-cell proteomic assays such as flow cytometry, mass cytometry (CyTOF), and imaging-based proteomics, single-cell metabolomics, singlecell secretome analyses, microfluidic or droplet-based single-cell partitioning assays, spatially resolved single-cell transcriptomic or proteomic assays, and multiomic workflows that measure combinations thereof within the same cell. In some aspects, as described here and throughout the specification, the compounds of this disclosure may be used in various methods and workflows for performing single-cell analytical assays. In one aspect, compounds of this disclosure may be used to prepare a population of cells to reduce intercellular adhesion and to modulate membrane permeability without inducing complete cellular lysis. In one aspect, compounds of this disclosure may be used in loading buffers or cell suspension buffers to maintain cell viability, control osmolarity, prevent clumping, and enable stable flow into microfluidic channels. In one aspect, compounds of this disclosure may be used to (i) maintain the integrity of individual cells or nuclei during physical or microfluidic isolation, (ii) minimize adsorption of biomolecules to assay surfaces, and / or (iii) stabilize enzymatic or amplification reactions performed downstream. In one aspect, compounds of this disclosure may be used to facilitate generation of uniform water-in-oil emulsions for droplet-based single-cell partitioning, enhance single-entity encapsulation efficiency, or enable controlled, selective permeabilization of plasma or nuclear membranes to permit extraction or detection of intracellular analytes. The incorporation of compounds of this disclosure into sample preparation buffers, loading buffers, cell suspensions buffers, mountants, fluidic buffers, reaction buffers, or partitioning buffers thereby improves assay reproducibility, reduces aggregation-associated capture artifacts, and enhances sensitivity in single-cell genomic, transcriptomic, proteomic, or epigenomic workflows.

[0242] Nonionic surfactants play a critical role in the performance and reliability of a wide range of fluidics instruments and automated systems used in biological, chemical, and analytical workflows, and are incorporated into many systems solutions. Because these surfactants lack an ionizable headgroup, they provide surface-active behavior without introducing charge-dependentinteractions that could interfere with sensitive assay chemistries or instrumentation materials. In fluid handling systems and other automated systems, nonionic surfactants are commonly employed to reduce surface tension and promote consistent wetting of tubing, channels, valves, cuvettes, syringes, and flow cells, thereby enabling more stable liquid transport and minimizing the formation of air bubbles that disrupt flow accuracy. Their ability to prevent nonspecific adsorption of biomolecules to internal surfaces, and thus minimize carryover, is particularly valuable in instruments designed for sample preparation, chromatography, cytometry, or nucleic acid analysis, where even minor analyte losses can compromise sensitivity and precision. In flow cytometry instruments, for example, nonionic surfactants are frequently incorporated into sheath fluids and maintenance solutions to improve fluidic stability and overall system performance. Low concentrations of these surfactants reduce surface tension within the fluidics pathway, thereby minimizing bubble formation, salt crystallization, and nozzle clogging that can adversely affect laser interrogation and signal acquisition. The chemical compatibility, low foaming characteristics, and broad tolerance to pH and ionic strength of nonionic surfactants make them particularly well suited for integration into complex, multi-step fluidics workflows. In automated analyzer systems, for example, nonionic surfactants are used to stabilize immunoassay reagents, particulate suspensions, chemiluminescent labels, and enzyme conjugates, enabling uniform mixing and improved assay kinetics to support reproducible reaction formation and optimal signal generation across a wide variety of automated diagnostic platforms. In some aspects, nonionic surfactants may also be used in mountants used in certain workflows. In some aspects, compounds of this disclosure may be used in the manufacturing and processing of mountants for use in various applications and workflows.

[0243] In addition to improving flow stability, nonionic surfactants are frequently used to condition fluidic pathways, enhance mixing efficiency, and maintain the cleanliness of internal components. They can act as dispersing agents for reagents or particles, stabilizers for biological samples, and lubricants for moving interfaces such as piston seals or microvalves. Many automated systems incorporate nonionic surfactants into wash buffers or priming solutions to remove residual reagents and prevent carryover between analytical runs. Nonionic surfactants can also be used in calibrators, controls, and standards to stabilize proteins, antibodies, enzymes, and labeled detection reagents during storage and deployment on automated systems, to aid in preserving known analyte concentrations and ensuring accurate calibration curves and qualitycontrol performance. In chemiluminescent immunoassays, nonionic surfactants may also be used in pre-trigger and trigger solutions. In pre-trigger solutions, nonionic surfactants facilitate solubilization of hydrophobic chemiluminescent labels, prevent precipitation following pHchanges, promote uniform wetting of the reaction cuvette, and reduce nonspecific adsorption of reagents prior to activation of the signal-generating chemistry. In trigger solutions, surfactants similarly enhance fluidic stability by improving reagent delivery and dispersion, minimizing bubble formation that interferes with optical detection, and maintaining the solubility and accessibility of signal-generating components during the rapid oxidation event. These actions enable consistent, high-intensity chemiluminescent output and improved analytical precision. Accordingly, the surfactants according to this disclosure can be used as an alternative to other non-ionic surfactants commonly used in the reagents provided herein.

[0244] In addition, nonionic surfactants such as those according to the present disclosure may be incorporated into cleaning solutions for all types of instruments to help limit sample carryover by promoting efficient rinsing of tubing and flow cells without introducing ionic species that might interfere with optical detection or cell viability.

[0245] In some aspects, compounds of this disclosure may be used with fluidics instruments and automated systems in the various manners described above, including priming and system conditioning solutions, trigger and pre-trigger solutions, sample transport or carrier fluids, reagent dilutions, assay buffers, calibrators, controls, standards, bead suspension and particle handling buffers, microfluidic chip fluids, sheath fluids, maintenance solutions, wash buffers and solutions, decontamination solutions, and cleaning solutions. One of skill in the art would readily appreciate the importance of nonionic surfactants in fluidics instruments and appreciate the importance of a strong nonionic surfactant, with an improved toxicological and environmental profile over surfactants such as Triton™ X- 100 and NP-40, in these types of applications.

[0246] Similar to fluidic instruments and automated systems, nonionic surfactants are used with robotic laboratory instruments and systems, which frequently handle small volumes through pipette tips, tubing, or microfluidic channels. Many particle types used in such systems, e.g., proteins, nanoparticles, extracellular vesicles (EVs), viral vectors, pigments, polymer beads, readily adsorb onto plastics or remain behind in dead volumes. Nonionic surfactants may be incorporated into robotic laboratory instruments and associated reagents and automated workflows and associated reagents to improve sample stability, transfer accuracy, and overall analytical reliability. In robotic liquid-handling systems, surfactants reduce particle adhesion to pipette tips, tubing, microfluidic channels, and labware, thereby minimizing sample loss and improving recovery across automated dilution, mixing, and dispensing steps. They further promote uniform dispersion of nanoparticles, proteins, extracellular vesicles, beads, and other particulate analytes during robotic preparation and transport, preventing aggregation or settlingthat would otherwise introduce variability. By reducing foaming, bubble formation, and surfacetension-related handling errors, nonionic surfactants enhance pipetting precision and reduce clogging or aspiration failures in high-throughput robotic platforms. Additionally, they lower carryover by improving cleaning efficiency in robotic wash paths and help stabilize particle-based reagents during extended deck residence times. Collectively, the use of nonionic surfactants in robotic workflows yields more reproducible, contamination-resistant, and instrument-compatible particle characterization, supporting robust QC and R& D applications. One of skill in the art would readily appreciate the importance of compounds of this disclosure as alternative nonionic surfactants in various robotic instruments and workflows.

[0247] Particle characterization is an essential component of quality control and research and development across pharmaceuticals, biologies, materials science, nanotechnology, environmental testing, food science, and chemical manufacturing. Conventional techniques such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), laser diffraction, coulter counting, flow cytometry, zeta potential analysis, microscopy, and chromatographic separations often suffer from particle aggregation, surface adsorption, inconsistent dispersion, foaming, and signal instability.

[0248] Nonionic surfactants provide broad utility across particle characterization workflows by improving dispersion, stability, and measurement accuracy without introducing charge-related artifacts. They can be used to stabilize particle suspension, reduce nonspecific adsorption of particles to surfaces, improve uniformity of replicate measurements, enhance sample uniformity, improve accuracy in particle counting assays, reduce particle aggregation in light-scattering assays such as DLS, MALS, NTA, and laser diffraction, enabling more reliable size and polydispersity measurements. Nonionic surfactants also prevent particle adhesion to cuvettes, tubing, and microfluidic channels, improving recovery and reducing fouling in instruments including flow cytometers, NTA systems, and impedance-based particle counters. By minimizing clumping and stabilizing heterogeneous particle types, such as extracellular vesicles, viral particles, protein complexes, polymer nanoparticles, and inorganic materials, they enhance particle counting and concentration measurements while preserving the integrity of delicate samples. In zeta potential assays, these surfactants stabilize dispersions without contributing ionic charge, supporting electrokinetic measurements that would otherwise be compromised by aggregation. Additionally, they improve uniform sample preparation for microscopy and spectroscopy, facilitate wetting and dispersion of hydrophobic powders, and support thermal, chromatographic, and automated high-throughputworkflows by reducing artifacts such as foaming, inconsistent handling, or drying-induced aggregation.

[0249] Across these diverse applications, the advantages of nonionic surfactants include improved reproducibility, reduced sample loss, enhanced instrument cleanliness, and broader compatibility with sensitive optical and electrokinetic detection methods. Their chemically neutral nature minimizes interference with measurement principles while still providing robust steric stabilization, making them particularly suited for regulated QC and R& D environments. Overall, nonionic surfactants offer a versatile, low-impact solution that enhances measurement precision and operational efficiency in virtually every stage of particle characterization. One of skill in the art would readily understand the advantages of the compounds of this disclosure and the various uses for such compounds in particle characterization workflows.

[0250] Compounds of this disclosure can be used in cleaning solutions and cleaning protocols used in multiple workflows, including cleaning solutions for a broad range of instruments.

[0251] Nonionic surfactants are widely used to improve the stability, functionality, and detection performance of detectable labels employed in assays, analytical workflows, biosensors, in-vitro diagnostic (IVD) devices, and other workflows, assays, and instruments described herein. Such detectable labels include fluorescent and chemiluminescent dyes, enzymatic and affinity tags, absorbance / color dyes (e.g., Coomassie, amine-reactive dye labels), near-IR emission dyes, radioisotopes, nanoparticle conjugates, mass-spectrometry labels, and time-resolved luminescent complexes. Nonionic surfactants are frequently incorporated into storage buffers and formulations for these labels used with protein and nucleic acid processes to enhance stability, prevent aggregation, reduce adsorption, and extend reagent shelf life.

[0252] In one aspect, nonionic surfactants are frequently incorporated in various assay reaction buffers involving labels because they improve solubility, prevent aggregation and denaturation, reduce nonspecific binding, enhance labeling efficiency without interfering with reaction chemistry, and reduce nonspecific adsorption to plastic, glass, or membrane surfaces. By providing steric hindrance and modulating hydrophobic interfaces around dyes, chemiluminescent moieties, enzyme labels, metal chelates, or nanoparticle surfaces, they help preserve structural integrity during storage and assay execution. Surfactants additionally enhance the solubilization and dispersion of hydrophobic or amphiphilic labels — such as near-IR fluorophores, acridinium esters, and plasmonic nanoparticles — ensuring uniform reagent distribution and consistent interaction with substrates, detection reagents, or excitation energy. Nonionic surfactants also improve assay specificity and reduce background byminimizing nonspecific binding in wash buffers, reaction mixtures, and incubation solutions. This function is critical in immunoassays, bead-based assays, lateral flow formats, spatial proteomics, and automated IVD platforms, where surface-mediated artifacts can compromise sensitivity and generate false positives. For enzyme-labeled polypeptides, such as HRP- or alkaline phosphatase-conjugated antibodies, nonionic surfactants maintain catalytic activity, prevent irreversible structural changes, and promote uniform substrate access. In chemiluminescent systems, they help maintain solubility and reactivity of hydrophobic luminophores, supporting consistent signal generation. Nanoparticle-based labels — including gold nanoparticles, quantum dots, magnetic beads, and upconverting nanoparticles — benefit from improved colloidal stability and reduced aggregation, enabling reliable optical or magnetic performance. Nonionic surfactants similarly enhance the behavior of affinity-tagged polypeptides (e.g., biotinylated proteins, His-tagged constructs, FLAG-tagged antibodies, and enzymatically biotinylated AviTag peptides) by maintaining tag accessibility and preventing oligomerization or nonspecific adherence. In mass-tagged systems, such as metal-chelate-labeled antibodies or isotopically encoded peptides for mass cytometry or quantitative proteomics, nonionic surfactants reduce precipitation and carryover, improving quantitative fidelity. In workflows that incorporate fluorescence and other staining processes used for various cell imaging techniques, nonionic surfactants reduce nonspecific staining by blocking hydrophobic interactions, disrupting weak off-target antibody binding, and improving wash efficiency — resulting in a cleaner, higher-contrast signal, and improving intracellular access and image quality.

[0253] Nonionic surfactants also serve as important excipients in multiple components in biological and histological staining and label kits. They modulate membrane permeability, improve reagent wetting, and reduce nonspecific interactions in staining workflows such as immunohistochemistry, immunocytochemistry, immunofluorescence, and flow cytometry. In permeabilization and wash buffers, they facilitate penetration of antibodies and dyes while lowering background. As wetting agents, they promote uniform coverage of tissue sections or cell suspensions and enhance stain penetration into dense or hydrophobic regions. Low surfactant concentrations also stabilize stain components, limit aggregation or precipitation, and improve lotto-lot reproducibility. These benefits extend to histological stains (e.g., trichrome, PAS, silver stains) and protein or nucleic acid staining kits (e.g., Coomassie, fluorescent gel stains, nuclear dyes), where surfactants support uniform stain distribution, reduce artifacts, and increase sensitivity and shelf life.

[0254] Accordingly, compounds of this disclosure may be incorporated into a wide range of label formulations and storage buffers, as well as in various biological, diagnostic, andhistological staining kit components, including label and stain formulations and storage buffers, permeabilization buffers, wash buffers, reaction mixtures, detection buffers, and incubation solutions.

[0255] Techniques such as gel electrophoresis are used to separate biomolecules, including proteins and nucleic acids, based on their molecular weight or size. For example, gel electrophoresis is commonly used to separate proteins, such as native proteins, by physical shape (e.g., structure), size and charge or denatured proteins by length of polypeptide. Separated biomolecules form individual bands in lanes of a gel. Following gel electrophoresis, a variety of dyes, stains or reagents can be used to detect and quantify bands of biomolecules that have been electrophoresed. Gel electrophoresis techniques useful for the analysis of proteins and nucleic acids include agarose and denaturing agarose electrophoresis for resolving DNA and RNA fragments, native and denaturing polyacrylamide gel electrophoresis (including SDS-PAGE) for separation of proteins or small nucleic acid species based on size or conformation, pulsed-field gel electrophoresis for fractionating high-molecular-weight DNA, and capillary or microfluidic electrophoresis systems for automated, high-resolution characterization of protein or nucleic acid samples.

[0256] As noted in other sections herein, cell lysates or permeabilized cell extrudates and materials obtained by cell lysis or cell permeabilization using compounds of the disclosure and formulations thereof, can be used for applications such as gel electrophoresis. In gel electrophoresis, a gel enclosed in a gel cassette comprising either capillaries, a glass / plastic tube, or more traditionally sandwiched as a slab between glass or plastic plates. At the top of a gel cassette there are sample wells created as impressions in the gel where a sample (such as a cell lysate or cell permeate or materials derived therefrom) can be loaded. Gels have an open molecular network structure, defining pores which are saturated with an electrically conductive buffered solution. These pores are large enough to admit passage of the migrating macromolecules through the gel. The gel cassette is placed in a chamber in contact with buffer solutions which make electrical contact between the gel and the cathode or anode of an electrical power supply. A sample containing the target analytes and a tracking dye is placed on top of the gel. An electric potential is applied to the gel causing the sample target analytes and tracking dye to migrate toward the bottom of the gel. The electrophoresis is halted just before the tracking dye reaches the end of the gel. The locations of the bands of separated target analytes are then determined. By comparing the distance moved by particular bands in comparison to the tracking dye and macromolecules of known size, the size and other details of the separated macromolecules can be determined.

[0257] Polyacrylamide gels, including but not limited Bis-Tris, Bis-Tris-Plus, Tris Glycine gel, and Tris Acetate, are commonly used for electrophoresis. Other gels suitable for electrophoresis include agarose gels and starch gels. Polyacrylamide gel electrophoresis or PAGE is popular because the gels are optically transparent, electrically neutral and can be made with a range of pore sizes.

[0258] Methods of making PAGE gels are known. See Hames and Rickwood, Gel Electrophoresis of Proteins (2d ed. Oxford University Press, 1990); Andrews, Electrophoresis (2nd ed. Oxford University Press, 1986). In general, concentrated stock solutions containing acrylamide monomer, a crosslinker such as bisacrylamide, gel buffers (e.g., Tris-glycine), and denaturing agents such as sodium dodecyl sulphate (“SDS”) are prepared. These stock solutions can be stored until a gel is needed. To manufacture a gel, the stock solutions are mixed with water in proportions according to the final desired concentrations of the various constituents. Polymerization of the gel is accomplished by adding an oxidizing agent polymerization initiator such as ammonium persulfate ((NH4)2(SO4)2; APS). Often a polymerization accelerator such as tetramethylethylenediamine (TEMED) is added to facilitate rapid polymerization.

[0259] In some cases, a stacking gel is used. A stacking gel is a gel poured on top of a resolving gel. The stacking gel typically has a lower pH (6.8 vs. 8.8) and a lower percentage of polyacrylamide (4% vs. 8-12%) than the resolving gel. The purpose of the stacking gel is to concentrate and focus the sample volume so that the molecules of interest (e.g., proteins) enter the resolving gel in a tight band having a volume much less than what was loaded.

[0260] In addition to uses for permeabilization of cells and initial solubilization of samples, nonionic surfactants, like the compounds of this disclosure, may be used in gel electrophoresis workflows for protein solubilization, membrane protein handling, and native (nondenaturing) electrophoresis. Nonionic surfactants serve important roles in gel electrophoresis workflows, particularly in native or non-denaturing formats. In contrast to SDS-PAGE, which relies on anionic surfactants to denature proteins and confer uniform negative charge, native electrophoresis techniques require the preservation of protein conformation, quaternary structure, and biological activity. Nonionic surfactants enable the extraction of target analytes, and the solubilization of proteins, multi-subunit complexes, and other hydrophobic species while largely maintaining their native structures. In native PAGE, Blue Native PAGE, and related separation methods, these surfactants improve sample solubility and compatibility with electrophoretic conditions without significantly altering intrinsic protein charge. Their inclusion can also reduce polypeptide aggregation, minimize adsorption of target analytes to sample-handlingsurfaces, and enhance reproducibility in electrophoretic separations. Accordingly, the surfactants of the present disclosure may be incorporated into gels, e.g. PAGE gels.

[0261] Despite their widespread use, existing workflows often face limitations, including incomplete solubilization of membrane proteins, loss of target antigenicity, suboptimal antibody penetration, inconsistent electrophoretic mobility, and unwanted background signal. The behavior of nonionic surfactants in these settings depends on multiple factors, including surfactant concentration, micelle formation, protein composition, and compatibility with downstream detection methods. As a result, there is an ongoing need for improved nonionic surfactants to enhance sample handling, sample preparation, immunodetection, and electrophoretic analysis while addressing the deficiencies of existing techniques. In some aspects, the compounds of this disclosure have been shown to provide advantageous use throughout gel electrophoresis workflows by providing such benefits, including improving protein stability, maintaining the native state of proteins, providing gentle solubilization, and preventing aggregation. For example, in some aspects, compounds of this disclosure may be used in sample buffers, loading buffers, or gel running buffers to prevent target analytes sticking to plastic or glassware, improve solubility and stability, improve reproducibility in low-concentration samples, and reduce streaking in native gels. In some aspects, compounds of this disclosure may be incorporated into loading buffers, along with a tracking dye, and optionally glycerol and / or a reducing agent. In some aspects, compounds of this disclosure may be incorporated into tracking dye formulations to prevent adsorption of dye components to container surfaces, improve solubility of hydrophobic additives, reduce precipitation of multi-component dyes, or enhance freeze-thaw tolerance. In other aspects, compounds of this disclosure may be incorporated into manufacturing and formulation processes for loading / tracking dyes to facilitate dispersion and wetting of dye components, stabilize emulsions or liquid dye concentrates, and enhance the long-term stability and uniformity of the resulting dye compositions. In other aspects, compounds of this disclosure may be used in transfer buffers, blocking buffers, wash buffers, or handling buffers used in gel electrophoresis.

[0262] In other aspects, the compounds of this disclosure may be used in various capillary electrophoresis (GE) techniques— including capillary gel electrophoresis, capillary zone electrophoresis, micellar electrokinetic capillary chromatography, capillary electrochromatography, capillary isoelectric focusing, and capillary isotachophoresis. — to reduce polypeptide adsorption to the capillary wall, stabilize electroosmotic flow, reduce polypeptide aggregation, maintain target analyte separation, stabilizepolypeptide membranes, modulate analyte flow, and solubilize polypeptides without denaturing them. In some aspects, the compounds of this disclosure may also be used to modify electroosmotic flow by transiently coating the capillary wall, alter selectivity through formation of micellar or pseudo-stationary phases, or facilitate the migration of amphipathic or membrane-associated molecules. One of ordinary skill in the art would be able to determine, through routine experimentation, the effective concentrations of the compounds of this disclosure in various formulations to improve resolution, peak symmetry, or reproducibility in capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, or other CE-based separation formats. In various implementations, the compounds of this disclosure may be selected to be compatible with the applied electric field, buffer chemistry, analyte class, and detection modality while maintaining the integrity of the separation environment.

[0263] Electrophoretically separated biomolecules are typically transferred from the separating gel onto another material in order to perform additional analysis on the biomolecules such as, but not limited to, immunological characterization, chemical reactions, quantitation, etc. Electro-blotting or electrotransfer is a method known in the art for transferring resolved or separated biomolecules from a gel onto another material.

[0264] Accordingly for further downstream analysis and applications, gel electrophoresis is often followed by electrophoretic transfer of the biomolecules onto a membrane where the biomolecules can be further analyzed by specific immunostaining and / or other specific labeling methods to aid detection, visualization and quantification of the biomolecules. Electrotransfer is achieved by typically building an electrotransfer stack. In some embodiments, the step of stack assembly comprises the steps of: using a gel or a matrix or a material having ions; placing and orienting a gel comprising resolved biomolecules (nucleic acids or proteins) with the wells toward the bottom side of the electrotransfer cassette over the gel / matrix / material having ions. The electrotransfer methods and stack assemblies may be for wet-transfer, dry transfer or semi-dry transfer. In some non-limiting examples of a dry transfer, the gel / matrix / material having ions can comprise an ion reservoir or conductive ions in the gel / matrix / material that are not a liquid buffer. The membrane can then be probed with analyte binding agents, e.g., antibodies (such as primary and / or secondary antibodies) to detect the proteins on the membranes. Probing can be done by a variety of methods such as sequential lateral flow (SLF) assays, for example, using Invitrogen ™ iBindTMWestern Systems.

[0265] Once probed, the membrane is also referred to as a Blot. Membrane or Blot imaging is used to visualize, detect and quantify biomolecules that are transferred ontomembranes. When proteins are transferred onto a membrane and probed with probes comprising one or more antibodies, the membrane with the protein-probe complexes is referred to as Western-blot. Typical exemplary membranes for protein transfer are membranes comprising nitrocellulose or polyvinylidene fluoride (PVDF). When DNA is transferred to a membrane and probed with probes, the membrane with the DNA-probe complex is referred to as a Southern-blot. When RNA is transferred to a membrane and probed with probes, the membrane with the RNA-probe complex is referred to as a Northern-blot.

[0266] Nonionic surfactants are widely employed throughout blotting and Western-blot workflows to enhance assay performance by modulating protein-surface and antibody-surface interactions without introducing additional charge. In contrast to ionic surfactants, nonionic surfactants such as Tween-20, Triton™ X-100, and NP-40 do not disrupt protein secondary structure but provide controlled reduction of hydrophobic interactions. According some embodiments of the present disclosure, one or more of the nonionic surfactants including Tween-20, Triton™ X-100, and NP-40 etc. will be replaced with compositions and formulations of the present disclosure. During Western blot processing, these agents are commonly incorporated into blocking buffers, transfer buffers, and wash buffers to minimize nonspecific antibody binding, stabilize protein-membrane associations, and improve signal-to-noise ratios. In some protocols, low concentrations of nonionic surfactants are also used in transfer or stripping buffers to facilitate more efficient protein migration or gentle removal of bound antibodies. Their mild, nondenaturing characteristics make nonionic surfactants indispensable for achieving reproducible, high-fidelity protein detection in immunoblotting applications.

[0267] In some aspects, compounds and formulations of this disclosure may be used in blotting and Western blotting processes, including in blocking buffers, wash buffers, transfer buffers, and stripping buffers. As demonstrated in the examples in this disclosure, the compounds of this disclosure have been shown not to disrupt the secondary structures of proteins.

[0268] Peptides and / or proteins may need to be quantitated to determine the amount of protein in a sample, such as samples generated by cell lysis by compounds and formulations of the disclosure. A variety of peptide quantification assay methods are known in the art. Commercially available colorimetric protein and peptide solution quantitation methods include biuret (Gornall et al. J. Biol. Chem. 177 (1949) 751 ), Lowry (Lowry et al. J. Biol. Chem. 193 (1951 ) 265), bicinchoninic acid (BCA) (Smith et al. Anal. Biochem. 150 (1985) 76), Coomassie Blue G-250 dye-binding (Bradford, Anal. Biochem. 72 (1976) 248), and colloidal gold (Stoscheck, Anal. Biochem. 160 (1987) 301). Another method combines the biuret reaction and the copper(1)-bathocuproine chelate reaction (Determination of Proteins by a Reverse Biuret Method Combined with the Copper-Bathocuproine Chelate Reaction. Clinics Chimica Acta., 216 (1993) 103-111).

[0269] The Lowry method is a modified biuret reaction. It occurs in two steps: first, peptide bonds react with copper(ll) ions under alkaline conditions, then Folin-Ciocalteau phosphomolybdic-phosphotungstic acid reduces to heteropolymolybdenum blue by copper-catalyzed oxidation of aromatic amino acids. The absorption maximum of the product is 750 nm. The Lowry method is more sensitive than the biuret method, with a linear sensitivity of 0.1 mg protein / ml to 1.5 mg protein / ml for bovine serum albumin (BSA). Certain amino acids, surfactants, lipids, sugars, and nucleic acids interfere with the reaction. The reaction is pH dependent and pH should be maintained between pH 10 and pH 10.5.

[0270] Another well known gold standard method in the art for quantification of proteins and peptides includes the BCA method (such as those by the Pierce™ BCA Protein Assay Kits). The BCA method is related to the Lowry method in that peptide bonds in proteins first reduce cupric ion (Cu2+) to produce a tetradentate-cuprous ion (Cu1+) complex in an alkaline medium. The cuprous ion complex then reacts with BCA (2 molecules BCA per Cu1+) to form an intense purple color that can be measured at 562 nm. The BCA-Copper reaction is shown below:(BCA) where salt

[0271]

[0272] Because BCA is stable in alkaline medium, the BCA method can be carried out in one step, compared to two steps needed in the Lowry method. In general, the BCA method better tolerates potential inhibitory or interfering compounds in the sample compared to the Lowry method. For example, up to 5% of each of sodium dodecyl sulfate (SDS), Triton™ X-100, and Tween-20 can be present and not interfere with the BCA method, compared to generally lower percentages of nonionic surfactants that are used with the Lowry method. The BCA method also may have increased sensitivity and an expanded linear working range compared to the Lowry method. A MICRO BCA™ Protein Assay Kit (Thermo FisherScientific) permits quantitation of dilute sample solutions (0.5 pg / ml to 20 pg / ml) by using larger sample volumes to obtain higher sensitivity.

[0273] In some aspects, the compounds of this disclosure may be used in various protein quantification assay workflows to improve the solubility, stability, and analytical recoverability of proteins within a given sample matrix. In some aspects, compounds of this disclosure may be used in assay formulations, sample preparation buffers, and assay-compatible sample dilution buffers to disperse hydrophobic or membrane-associated proteins, reduce aggregation, and minimize adsorption of proteins to assay surfaces. In some aspects, the compounds of this disclosure may be incorporated into dyes and dye reagents, such as Coomassie dyes; they may also be used in standard buffers or standard curve preparation buffers, in blocking or anti-adsorption buffers, in fluorescent or dye-based as assay buffers, and in assay working buffers. The presence of these compounds may facilitate uniform protein dispersion, mitigate matrix-related interference, and enable reliable quantification of proteins that are otherwise prone to precipitation, denaturation, or loss due to surface binding or sample handling artifacts.

[0274] Immunoassays may be configured as competitive, noncompetitive, homogeneous, or heterogeneous formats and may employ labeled or unlabeled antibodies or antigens. They may be configured to detect, identify, or quantify a single target, or multiple targets in a single sample (multiplex). Suitable detection modalities include, without limitation, colorimetric, fluorescent, chemiluminescent, enzymatic, electrochemical, or particle-based readouts. Immunoassays may be performed in various platforms such as ELISA, chemiluminescent immunoassays, radioimmunoassays, lateral flow devices (including rapid, point of care immunoassays), bead-based assays, and automated clinical analyzers, and are applicable to the detection of proteins, peptides, small molecules, nucleic acids, metabolites, or other analytes capable of antibody recognition. Other types of immunoassays include flow cytometry-based immunoassays for cell surface or intracellular markers; immunoprecipitation techniques (IP, CoIP, ChIP) for isolating proteins or protein-nucleic acid complexes; agglutination assays such as latex particle tests and blood typing; and functional immunoassays like antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and viral neutralization assays that measure antibody activity.

[0275] Immunoassay based protein detection methods such as ELISAs, are well known in the art. Proteins from samples generated by cell lysis or cell permeabilization by compounds and formulations of the disclosure, can be further analyzed by a variety of immunoassay methods including ELISA, ProcartaPlexTMassays, ProQuantum ™ Assays and the like. ProcartaPlex assays use xMAP technology to measure up to 80 different protein analytessimultaneously per well. Capture antibodies are bound to LuminexTMbeads which are internally dyed. The conjugation of a specific antibody to a distinct bead allows for analysis of multiple analytes in a single well. Samples are mixed with the bead sets. Analytes of interest within the sample are bound by the capture antibodies. Fluorescently labeled detection antibodies specific to the analytes of interest are added, forming an antibody-antigen sandwich. Completed assays are read on a LuminexTMinstrument where one laser classifies the bead type to determine the analyte that is being detected while a second laser determines the magnitude of the bound analyte (PE-derived signal).

[0276] In some aspects, the compounds may be incorporated into one or more components of a kit. In one aspect, compounds may be incorporated into one or more components of a kit having (1) a capture analayte reagent, wherein the capture analyte reagent comprises a bead or microcarrier conjugated to a first analyte binding agent. The analyte binding agent can be an antibody or fragment thereof, or a non-antibody capture agent, in either case, which specifically binds a first epitope on a target analyte; (2) a detection analyte reagent, wherein the detection analyte reagent comprises a detectable label. The analyte binding agent can be an antibody or fragment thereof, or a non-antibody capture agent, in either case, which specifically binds a second epitope on the target analyte; and (3) optionally one or both of a lysis buffer and a wash buffer; where the compounds of this disclosure can be incorporated into one or more of the capture analyte reagent, the detection analyte reagent, the lysis buffer, or the wash buffer. In one aspect, the capture analyte reagent comprises a bead or microcarrier that is internally labeled with a dye. In one aspect, the disclosed kit may also comprise a plurality of capture analyte binding reagent / detection analyte reagent pairs, wherein each pair specifically binds to a first and second epitope on a unique target analyte respectively. In one aspect, the disclosed kit may comprise 2 to 100 capture analyte binding reagent / detection analyte reagent pairs.

[0277] Invitrogen ™ ProQuantumTMimmunoassays are easy-to-run, high-performance, target-specific protein detection assays. Utilizing proximity-based amplification technology, ProQuantum assays offer the analyte specificity of antibody-antigen binding with the signal detection and amplification capabilities of qPCR to achieve a highly sensitive assay. ProQuantum assays can also typically detect lower levels of protein with lower sample consumption than traditional methods. In some embodiments, non¬ ionic surfactants used in Invitrogen ™ ProQuantum ™ immunoassays can be replaced with compounds and formulations of the present disclosure.

[0278] In one aspect, compounds of this disclosure may be used in a kit comprising: (1) an analyte capture reagent / analyte detection reagent pair, wherein the analyte capture agent comprises an analyte binding agent a first oligonucleotide probe, wherein the analyte binding agent specifically binds to a first epitope on a target analyte; (2) an analyte detection agent comprising analyte binding agent and a second oligonucleotide probe, wherein the analyte binding agent specifically binds to a second epitope on the target analyte; (3) optionally, a splint oligonucleotide probe that is complementary to at least a portion of the first oligonucleotide probe and at least a portion of the second oligonucleotide probe; and (4) optionally, one or more of a lysis buffer, a DNA ligase, a DNA polymerase, dNTPs, and an amplification buffer; wherein one or more of the analyte capture reagent, analyte detection reagent, or lysis buffer comprises a surfactant according to compounds of this disclosure.

[0279] In one aspect, compounds of this disclosure may be used in a method to detect one or more target analytes from a sample comprising: (1 ) contacting the sample comprising one or more cells with an amount of a composition comprising a compound of this disclosure effective to facilitate permeabilization of the one or more cells in the sample to obtain a cell lysate comprising the one or more analytes; (2) contacting the cell-lysate with: (a) an analyte capture reagent / analyte detection reagent pair, wherein the analyte capture agent comprises an analyte binding agent and a first oligonucleotide probe, wherein the analyte binding agent specifically binds to a first epitope on a target analyte; (b) an analyte detection agent comprising analyte binding agent and a second oligonucleotide probe, wherein the analyte detection agent specifically binds to a second epitope on the target analyte; and (c) a splint oligonucleotide probe that is complementary to at least a portion of the first oligonucleotide probe and at least a portion of the second oligonucleotide probe; (d) a DNA ligase; wherein one or more of the analyte capture reagent, analyte detection reagent, or lysis buffer comprises a compound of this disclosure; (3) ligating the first and the second oligonucleotides under effective ligation conditions to form a ligation complex comprising the ligated oligos and the analyte capture reagent / analyte detection reagent pair; (4) optionally washing the ligation complex with a buffer that comprises a compound of this disclosure; (5) contacting the ligation complex with a polymerase chain reaction (PCR) master mix comprising: a DNA polymerase; optionally, NTPs; amplification buffer; and optionally, one or more wash buffer; to form a PCR complex; (6) subjecting the PCR complex to a polymerase chain reaction amplification reaction to amplify the ligated oligo, thereby amplifying the signal of the target analyte; and (7) detecting the amplification and thereby detecting the target analyte in the sample. In one aspect, the foregoing method is performed where the target analyte is found at low levels in the sample.

[0280] In some aspects, compounds of this disclosure can be used in various in vitro diagnostic (IVD) immunoassays, such as drug immunoassays, to enhance assay stability, specificity, and analytical performance across a variety of assay formats, including competitive immunoassays for small-molecule drugs, sandwich immunoassays for drug-induced biomarkers, anti-drug antibody (ADA) and neutralizing antibody assays for biologic therapeutics, and immunoassay-based drug-of-abuse screens. Nonionic surfactants may be included in sample pretreatment buffers, capture and detection reagent formulations, conjugate storage buffers, and wash solutions to improve the solubilization and dispersion of hydrophobic drug analytes or metabolites, prevent aggregation or denaturation of labeled antibodies, and reduce nonspecific binding or surface adsorption of assay components. Their presence maintains the structural and functional integrity of enzyme-, chemiluminescent-, fluorescent-, or nanoparticle-labeled immunoreagents and supports consistent antibody-drug interactions during competitive or noncompetitive binding reactions. As a result, the use of nonionic surfactants provides improved background reduction, enhanced signal fidelity, and increased reproducibility in drug IVD immunoassays across diverse analytical platforms, including ELISA, chemiluminescent immunoassays, lateral flow devices, bead-based assays, and automated clinical analyzers.

[0281] In some aspects, compounds of this disclosure may be incorporated into many immunoassay systems to improve assay performance by modulating surface interactions, facilitating controlled removal of unbound components, stabilizing biomolecular reagents, and enhancing signal fidelity without substantially denaturing biomolecules such as antigens or antibodies. In some aspects, compounds of this disclosure may be included in sample pretreatment buffers, capture and detection reagent formulations, immunoassay reaction buffers, conjugate storage buffers, wash buffers, blocking solutions, reagent stabilizers and formulations, and sample diluents used in immunoassays.

[0282] In some aspects, in both solid-phase immunoassays, such as enzyme-linked immunosorbent assays and immunoprecipitation assays, and flow-based formats, including lateral-flow and bead-based assays, compounds of this disclosure may be used to maintain protein solubility, stabilize enzyme conjugates, minimize background signal, and support reproducible antigen-antibody interactions and bead-antibody interactions. In some aspects, compounds of this disclosure may be used in immunoassay workflows to reduce undesired adsorption of proteins to plastics, nitrocellulose, or latex particles. In some aspects, compounds of this disclosure may be used during sample or analyte binding agent (e.g., antibody) incubation to reduce nonspecific interactions, improve uniform wetting of wells, and stabilize sensitive proteins. In some aspects, for example in automated immunoassay platforms, compounds of thisdisclosure may be used in wash solutions, reagent stabilizers, probe rinse solutions, and other cleaning solutions to stabilize analytes during storage or automated processing, reduce instrument carryover, and preserve reagent integrity during storage and dispensing. In some aspects, for example in IVD immunoassays, compounds of this disclosure may be included in sample pretreatment buffers, capture and detection reagent formulations, conjugate storage buffers, and wash solutions. In some aspects, for example in immunofluorescence assays, nonionic surfactants compounds of this disclosure may be used in permeabilization buffers and blocking buffers; in wash solutions between antibody incubations to remove unbound antibodies, aggregates, and background staining; and to aid in preserving the cell and epitope integrity, including by preserving target analyte structure, maintaining fluorescence of dyes, and preventing denaturation of target analytes.

[0283] Pull-down assays are affinity-based biochemical methods used to determine a physical interaction between two or more proteins. Pull-down assays are useful for both confirming the existence of a protein-protein interaction predicted by other research techniques (e.g., co-immunoprecipitation) and as an initial screening assay for identifying previously unknown protein-protein interactions. Pull down assays are used to isolate a target molecule (“bait”) and one or more interacting molecules (“prey”) from a complex mixture. In such assays, the bait molecule is immobilized on a solid support and exposed to a biological sample under conditions that permit specific binding. Interacting molecules are retained on the support, while non-binding components are removed through washing steps. The captured bait and prey proteins can then be released from the affinity matrix using an eluent buffer designed to disrupt the specific binding interaction. The retained molecules may subsequently be eluted and analyzed by techniques such as SDS-PAGE, immunoblotting, or mass spectrometry. Representative pull-down formats include, but are not limited to GST pull-down assays, wherein a glutathione S-transferase-tagged bait protein is immobilized on glutathione affinity resin; His-tag pull-down assays, wherein a polyhistidine-tagged bait protein is captured using nickel-nitrilotriacetic acid (Ni-NTA) or cobalt chelate resins; epitope tag pull-down assays, such as FLAG-, HA-, or Myc-tag pull-downs, in which an epitope-tagged bait is captured using beads bearing antibodies specific to the epitope; biotin-streptavidin pull-down assays, in which a biotinylated bait is immobilized on streptavidin-coated supports; nucleotide-dependent pulldowns, including GTP- or GDP-loaded affinity matrices for isolating active or inactive forms of small GTPases; polypeptide, DNA, or RNA pull-down assays, wherein immobilized polypeptides or nucleic acids capture interacting proteins or protein complexes; and affinity resin pull-downsutilizing natural ligand interactions, such as calmodulin resin for calmodulin-binding proteins or lectin-based resins for glycoproteins.

[0284] Compounds of this disclosure may be incorporated into pull-down assay buffers to facilitate extraction, solubilization, and stabilization of proteins while maintaining native conformations and interaction states. Compounds of this disclosure can be incorporated into lysis buffers, to disrupt cellular membranes without denaturing proteins or impairing binding interactions between the bait and prey molecules; they can be incorporated in wash buffers, including to reduce nonspecific adsorption of proteins to the solid support, thereby improving assay specificity and signal-to-noise ratio; and they can be used in eluent buffers, including to maintain solubility, reduce nonspecific binding, and stabilize protein complexes during elution. The concentration and identity of the surfactant may be selected to preserve fragile or transient protein complexes while minimizing background binding.

[0285] Gene expression analysis simultaneously compares the RNA expression levels of multiple genes (profiling) and / or multiple samples (screening). This analysis can help scientists identify the molecular basis of phenotypic differences and to select gene expression targets for in-depth study.

[0286] Such gene expression differences can lead to potential biomarker discovery for a particular disease phenotype and enable further biomarker validation. Gene expression analysis provides valuable insight into the role of differential gene expression in normal biological and disease processes and in the identification and verification of biomarker signatures.

[0287] Analysis of gene expression may be done downstream in a sample comprising cells that are lysed or permeabilized by compounds and formulations of the disclosure. One such assay is the InvitrogenTMQuantiGeneTMAssays that provide an accurate and precise method for single or multiplexed gene expression quantitation,

[0288] QuantiGene Plex and SinglePlex assays utilize branched DNA (bDNA) technology. bDNA technology utilizes sequential nucleic acid hybridization for a unique approach to RNA and DNA quantification by amplifying a reporter signal rather than the template. This measures the transcripts at physiological levels.

[0289] First, cells are lysed using cell lysis and permeabilization methods with compositions of the present disclosure to release the target RNA or DNA. Alternatively, tissue samples can be homogenized by subjecting tissues to compositions and compounds of the disclosure to release the target RNA or DNA. Second, an oligonucleotide probe set is incubated with the target RNA or DNA. During this incubation, the probes cooperatively hybridize to the target. Third, signal amplification is performed via sequential hybridization of the bDNA pre-amplifier, amplifier, and labeled probe molecules to the target. Addition of a chemiluminescent substrate (singleplex assays) or fluorescent reporter (multiplex assays) generates a signal directly proportional to the amount of target RNA or DNA present in the sample.

[0290] The compounds of this disclosure may be incorporated into gene expression assay workflows and associated reagents to enhance nucleic acid recovery, improve reaction performance, and reduce analyte or reagent losses attributable to surface adsorption or aggregation. Lysis, extraction, reverse transcription, amplification, hybridization, or library preparation buffers may comprise one or more compounds of this disclosure. These nonionic surfactants may facilitate cellular disruption and solubilization of membrane components during nucleic acid isolation, stabilize enzymes such as reverse transcriptases and polymerases, improve homogeneity and wetting of reaction mixtures, reduce nonspecific binding in hybridization-based assays, and maintain the integrity of low-concentration RNA or cDNA samples. In various implementations, the compounds are present at concentrations selected to preserve compatibility with downstream enzymatic or hybridization chemistries while providing improved assay sensitivity, reproducibility, and quantitative accuracy across various formats workflows, including qPCR, RT-PCR, in situ hybridization, microarray, or sequencing-based gene expression formats.

[0291] Lateral flow assays or devices (LFAs) have an extensive history of use in both clinical and home settings, particularly in medical diagnostics. LFAs are simple to use and provide the ability to test for a variety of targets, such as biomarkers, proteins, hormones, drugs, and plasma components. LFAs are also used in food and environmental contexts to detect such things as pesticides, toxins, and allergens. LFAs rely on capillary transport of a biological sample across a porous membrane to enable specific binding interactions between one or more reagents and one or more target analytes. The presence of the target is then determined by measuring the presence of a label bound to the reagent.

[0292] An LFA may include a lateral flow strip with a sample loading section or pad; a conjugate section or pad; and an analysis section. There are multiple types of LFAs, including competitive LFAs such as those used to detect small molecule drugs of abuse and therapeutic drugs; sandwich LFAs, which detect macromolecules such as proteins, glycoproteins, and lipopolysaccharides; serological assays, which detect antibodies to a specific antigen in a biological sample; enzymatic or catalytic LFAs, which use enzymes to amplify signals at the test line; and nucleic acid LFAs which can be used in viral or bacterial detection. LFAs may be sequential, where sample, buffers and reagents flow in a controlled order, or non-sequential, where the entire sample flows continuously. Some LFAs, for example electrochemical lateral flowassays, may use an electrochemical detection, while others may use colorimetric detection, fluorescent detection, magnetic detection, or surface-enhanced Raman Spectroscopy detection.

[0293] To support consistent flow and reliable signal generation, LFAs routinely incorporate nonionic surfactants, for example, in sample application pads, extraction buffers, wash buffers, running buffers (or chase buffers, extraction buffers, assay buffers, or sample buffers), conjugate pads, and membrane blocking formulations to improve wetting, reduce nonspecific adsorption, disperse viscous sample components, and stabilize antibody-particle conjugates. Nonionic surfactants are also used in the production of cellulose membranes used in LFAs.

[0294] Despite their widespread use, existing nonionic surfactants present several limitations. Many nonionic surfactants exhibit insufficient compatibility with certain biological matrices, leading to variable flow rates or incomplete conjugate release. Some surfactants destabilize sensitive polypeptides or interfere with antigen-antibody binding at concentrations required to achieve adequate wetting. Others show limited effectiveness in solubilizing mucus or lipid-rich samples, such as those encountered in respiratory or environmental assays. Regulatory and environmental concerns have further restricted the use of certain surfactants.

[0295] In some aspects, compounds of this disclosure may be used advantageously in LFAs by incorporating the compounds into one or more of sample application pads, running buffers (also referred to as chase buffers, extraction buffers, assay buffers, or sample buffers), wash buffers or other reagents, conjugate pads, and membrane blocking formulations resulting in enhanced analyte accessibility, reduced nonspecific interactions, and superior compatibility with diverse sample types, while maintaining target stability and membrane integrity across all LFA components. In one aspect, compounds of this disclosure may be incorporated in a running buffer in an amount effective to improve capillary flow of the sample through a lateral flow strip.

[0296] In one aspect, compounds of this disclosure may be used in a sample application pad for use in a lateral flow assay device, where the sample application pad comprises a porous substrate configured to receive a liquid biological sample and to regulate the flow of the sample into a downstream conjugation zone; a treatment composition disposed on or within the porous substrate, the treatment composition comprising consisting essentially of, or consisting of: a nonionic surfactant incorporating compounds of this disclosure in an amount effective to enhance sample wetting and promote release of a target analyte from the biological sample (e.g., in an amount between 0.001% and 5%); and optionally one or more of a buffer, a blocking agent, anda stabilizer; wherein the sample application pad is configured such that, upon application of the biological sample, the treated porous substrate improves capillary flow uniformity of the sample, and delivers the sample to the downstream region of the lateral flow assay device. In some aspects, compounds of this disclosure may be used as a surface-active ingredient in the manufacturing of mixed cellulose ester membranes used in lateral flow assays and lateral flow strips, as well as any of the many products incorporating a microfiltration membrane. In some aspects, compounds of this disclosure may be used in the manufacturing of lateral flow assay devices and kits comprising the same.

[0297] In one aspect, compounds of this disclosure may be use in a lateral flow assay device for detecting a target analyte in a biological sample, the device comprising: (1) a sample application pad as described above; (2) a conjugate pad in fluid communication with the sample pad, wherein the conjugate pad contains a dried detection reagent comprising an analyte binding agent that comprises a detectable label and that specifically binds a first epitope on the target analyte forming a detection reagent-target analyte complex, when the target analyte is present, wherein the label selected from the group consisting of colloidal gold, latex particles, enzymes, and fluorescent dyes; (3) a porous membrane comprising: a test zone having an immobilized capture binding member configured to specifically bind the target analyte-detection reagent complex; (4) a control zone having an immobilized control binding member configured to excess detection reagent that is not bound to the target analyte; (5) an absorbent pad positioned downstream of the porous membrane and configured to draw the sample fluid through the device via capillary action; and (6) a housing that supports the sample pad, conjugate pad, membrane, and absorbent pad in a linear flow path; where, upon application of the biological sample to the sample pad, the sample flows laterally through the device, rehydrates the detection reagent, and wherein the detection reagent-target analyte complex produces a visually or instrumentally detectable signal in the test zone indicative of the presence of the target analyte in the biological sample.

[0298] In certain aspects, compounds of this disclosure may be used in compositions and methods for inactivating one or more viruses in virus-containing compositions using compounds of this disclosure. In other aspects, compounds of this disclosure may be used in compositions and methods of reducing viral contamination in a biological or biopharmaceutical composition, the method comprising treating the composition with a compound of this disclosure, under conditions effective to render the virus non-infectious. These compositions and methods are applicable in biological, pharmaceutical, or bioprocessing contexts where a virus may be present in a liquid medium that also comprises aproduct of interest, such as a recombinant protein, monoclonal antibody, or plasma-derived therapeutic. Inactivation is achieved by contacting the virus or virus-containing composition with a compound of this disclosure under conditions sufficient to reduce or eliminate the infectivity of the virus.

[0299] It is understood that conditions sufficient to inactivate the virus may refer to treatment conditions — such as a combination of surfactant concentration, contact time, and temperature — that result in the virus becoming non-infectious. In some embodiments, this means a reduction in viral infectivity of at least 4 log10, as determined using a validated assay such as a TCID50assay, plaque assay, or immunofluorescence-based readout in susceptible host cells. Such conditions may include, for example, treatment with a compound of this disclosure at a concentration of about 0.1% to 1% (w / v) for a period of about 30 to 60 minutes at a temperature of 20 °C to 40 °C, depending on the virus and matrix.

[0300] As described, the virus-containing composition may be any biological or biopharmaceutical material that comprises a virus or is suspected of comprising a virus. Representative compositions include, without limitation, mammalian or insect cell culture supernatants, clarified cell harvests, chromatography eluates, ultrafiltration / diafiltration fractions, and plasma-derived intermediates. In certain aspects, the composition comprises a process stream from the production of a recombinant therapeutic protein or monoclonal antibody. In other aspects, the virus-containing composition comprises a plasma fraction such as Cohn Fraction II or III, a cryoprecipitate, or a formulated immunoglobulin or albumin preparation. The methods disclosed herein are compatible with a wide range of matrices and do not substantially interfere with the integrity or recovery of the target biological product.

[0301] It is appreciated that compounds herein described may be applied to virus inactivation workflows across a broad range of biologically derived materials and therapeutic products, including but not limited to: plasma-derived medicines (e.g., immunoglobulins, albumin, clotting factors), recombinant biologies (e.g., monoclonal antibodies, enzymes, cytokines), gene therapy products (e.g., adeno-associated virus and lentiviral vectors), and cell therapy compositions (e.g., CAR-T cells, mesenchymal stem cells). The disclosed surfactant compounds may also be used to treat vaccine intermediates or finished vaccines (e.g., inactivated polio vaccine, toxoid formulations), as well as human- or animal-derived raw materials (e.g., fetal bovine serum, serum-derived trypsin, and other supplements) and diagnostic components that include human biological material (e.g., control samples, calibrators). In each of these categories, virus inactivation may be required by regulatory guidance, implemented as part of a standard process, or applied to reduce the risk of adventitious agent contamination in upstream materialsor downstream formulations. The disclosed methods thus provide a flexible platform for surf actant- mediated virus inactivation across therapeutic, research, and diagnostic use cases.

[0302] Surfactants are used extensively in the manufacture of active pharmaceutical ingredients, biopharmaceuticals, plasma or cell-derived products and cell and gene therapies. For example, non-ionic surfactants may be used in processes involving viral inactivation, purification, contaminant removal, post-production cleaning, and quality control testing, thereby contributing to improved product quality, process efficiency, and regulatory compliance with established viral safety and product integrity standards. In another aspect, they may be used in the capture process for non-enveloped viruses, such as adenovirus (AdV) adeno-associated virus (AAV), and the like, used in gene therapy. In vaccine development and production, non-ionic surfactants are often used in the virus splitting and inactivation steps.

[0303] It is further appreciated that the compounds described herein, including non-ionic surfactants, are commonly used in the production of recombinant proteins, antibodies, viral vectors, or other biological therapeutics, particularly in processes that employ human or animal cell lines. In these contexts, gentle surfactants are particularly desirable in order to effectively eliminate contaminants while maintaining the structural integrity, biological activity, and functional properties of the desired product. They may be used in a variety of upstream and downstream purification processes in the production of biologic agents, including viruses, antibodies, and vaccines. Non-ionic surfactants may be used in such processes to remove liquid culture medium from cells, or in cell lysis applications to release intracellular components. They may also be used in various recovery, purification or isolation processes to remove or inactivate unwanted contaminants such as adventitious viruses or host cell proteins, lipids, and other impurities. Such processes include a variety of biochemical techniques including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, hydrophobic interaction chromatography, or anion exchange chromatography) and filtration (e.g., molecular weight cut-off filtration, depth filtration using porous filter medium, or ultrafiltration).

[0304] In some aspects, the virus to be inactivated belongs to a virus family for which validated inactivation steps are required in the production of biopharmaceuticals or plasma-derived products. Virus families relevant to such applications include, but are not limited to, Retroviridae, Rhabdoviridae, Orthomyxoviridae, Paramyxoviridae, Flaviviridae, Coronaviridae, Herpesviridae, Togaviridae, and Filoviridae, as well as Adenoviridae, Parvoviridae, Reoviridae, and Picornaviridae. These families collectively include both viruses that possess lipid membranes and those that do not. Representative examplesinclude Xenotropic Murine Leukemia Virus (XMuLV) and Human Immunodeficiency Virus (HIV) (Retroviridae); Vesicular Stomatitis Virus (VSV) (Rhabdoviridae); Influenza virus (Orthomyxoviridae); Respiratory Syncytial Virus (RSV) and Parainfluenza viruses (Paramyxoviridae); Dengue virus, Zika virus, and West Nile virus (Flaviviridae); SARS-CoV-2 (Coronaviridae); Herpes Simplex Virus (HSV) and Cytomegalovirus (CMV) (Herpesviridae); Adenovirus type 5 (AdV5) (Adenoviridae); Minute Virus of Mice (MVM) (Parvoviridae); and Reovirus and Poliovirus (Reoviridae and Picornaviridae, respectively). The surfactant compounds of the present disclosure have demonstrated inactivation activity against multiple viruses from these families, making them suitable for use in validated virus clearance protocols required by regulatory agencies for biological products. Accordingly, the disclosed compounds can be used in processes targeting a broad spectrum of virus types, including both membrane-associated and non-membranous viruses, depending on the specific conditions and formulations employed.

[0305] The contacting step may be carried out by mixing the virus-containing composition with a compound described herein to achieve a final concentration of the surfactant ranging from about 0.01% to about 5% (w / v), such as 0.1% to 1.5%. The contact time may range from about 10 minutes to about 2 hours, and the temperature may range from about 20°C to about 40°C. In a representative embodiment, a clarified CHO cell harvest containing monoclonal antibody and potentially retroviral particles is treated with 1% (w / v) of a compound of this disclosure having a PEG-based hydrophilic group and a C9-C11 alkyl tail. The mixture is incubated at 25°C for 60 minutes under gentle agitation. Following treatment, viral infectivity may be assessed using standard cell-based (such as, TCID50) assays, and inactivation efficacy is confirmed when virus titers fall below detection limits or are reduced by at least 4 log10.

[0306] In additional embodiments, the virus-containing composition comprises a plasma-derived product. For example, the preparation of intravenous immunoglobulin (IVIG) in aqueous glycine buffer may be treated with a compound / surfactant described herein at a concentration of 1% to 2%, preferably 0.3% to 0.6%, for 4 hours at 30°C. This procedure can inactivate enveloped viruses such as HIV and HCV while preserving the structural and functional integrity of the immunoglobulin proteins. The surfactant may be removed by subsequent processing steps such as chromatography or ultrafiltration. In certain embodiments, the disclosed methods may be applied during early stages of plasma processing, for example in pooled plasma or cryo-poor plasma prior to fractionation, to reduce viral load at the bulk level.

[0307] It is understood that the method(s) disclosed herein are referred to as surfactant-mediated virus inactivation methods, wherein compound described herein functions as a nonionic surfactant that disrupts the lipid membranes or structural integrity of the virus, rendering itnon-infectious. Compounds of this disclosure, for example, provide an environmentally safer alternative to conventional surfactants such as Triton™ X-100 and do not release persistent alkylphenol degradation products. In some aspects, a compound of this disclosure replaces Triton™ X-100, or other nonionic surfactants that produces environmentally persistent or endocrine-disrupting degradation products, directly in standard virus inactivation protocols used in biologies manufacturing.

[0308] In certain aspects, the virus-containing composition may further comprise an aqueous buffer selected from phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane (Tris) buffer, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) buffer, citrate buffer, or acetate buffer. A compound of this disclosure may be formulated in such buffers, optionally with stabilizing excipients such as glycerol, and added directly to the virus-containing composition to initiate the inactivation process.

[0309] In some aspects, following the inactivation step, the compound may be removed or diluted to a residual concentration below 0.01% (w / v) prior to downstream purification steps, such as affinity chromatography, viral filtration, or formulation. In some embodiments, the method includes confirmation that the virus has been inactivated using validated infectivity assays or molecular detection techniques. In other embodiments, the method is implemented as a continuous inactivation step within an integrated downstream processing line.

[0310] These and other embodiments described herein provide robust, scalable, and regulatorily compliant methods for viral safety assurance in the manufacture of therapeutic proteins, plasma-derived biologies, and viral vector-based vaccines. Results of virus inactivation are shown below in tables, which describe the inactivation of a selected virus under specific conditions. It is appreciated that the inactivation efficacy may be assessed using standard infectivity assays (e.g., TCID50).

[0311] In one aspect, the disclosed compounds are used in methods for facilitating cell lysis, wherein the method comprises contacting a cell with a compound described herein in an amount effective, about 0.01% to about 5% (w / v), to disrupt cellular membranes and release intracellular components. In another aspect, the disclosed compounds are used in methods for virus inactivation, i.e., and particularly viruses that do not possess or have lipid membranes (nonenveloped). In most cases, the compounds are effective against lipid-enveloped viruses, which are susceptible to disruption by the amphiphilic structure of the surfactants. In certain embodiments, the method comprises combining a compound disclosed herein with a liquid composition comprising the virus, under conditions sufficient to render the virus non-infectious. Also disclosed herein are aspects of a kit comprising a compound of this disclosure and, in someembodiments, instructions or buffer components suitable for use in virus inactivation or cell lysis workflows.

[0312] In one aspect is a method of inactivating a virus, the method comprising contacting a virus or a virus-containing composition with a compound of this disclosure wherein the contacting is performed under conditions sufficient to inactivate the virus. In another aspect is the use of a compound of this disclosure for inactivating a virus in a virus-containing composition.

[0313] In yet another aspect, a kit is provided, for use in inactivating a virus in a viruscontaining composition, the kit comprising: (a) a compound of this disclosure; and (b) instructions for use, wherein the instructions describe contacting the compound with a virus or virus-containing composition under conditions sufficient to inactivate the virus. In some embodiments, the kit further comprises one or more buffers, excipients, or dilution components suitable for formulation or processing.

[0314] In another aspect, a composition is provided, comprising, consisting essentially of, or consisting of, a compound of this disclosure, and one or more additional components selected from a buffer, stabilizer, salt, or excipient, wherein the composition is suitable for use in a virus inactivation step. In some aspects, the buffer comprises one or more selected from citrate buffers (saline-sodium citrate (SSC)), phosphate buffers (phosphate buffered saline (PBS)), tris (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-1,3-diol)-based buffers, TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid)-based buffers, HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid)-based buffers, Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid)-based buffers, tricine (N-[tris(hydroxymethyl)methyl]glycine)-based buffers, TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)-based buffers, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)-based buffers, MES (2-(N-morpholino)ethanesulfonic acid)-based buffers, PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid))-based buffers, Cacodylate (dimethylarsenic acid)-based buffers, or any combination thereof. In some aspects, the stabilizer comprises glycerol. In certain aspects, the composition is provided as a concentrate, reconstitutable powder, or singleuse solution for use in bioprocessing or diagnostic applications. In some aspects, this composition further contains a virus.

[0315] In some aspects, the virus-containing composition contains a virus disclosed herein. In some aspects the virus-containing composition comprises a biopharmaceutical process stream, including but not limited to a cell culture harvest, clarified harvest, chromatography eluate, filtration retentate, or ultrafiltration / diafiltration (UF / DF) fraction. In some aspects the virus-containing composition comprises a recombinant protein ormonoclonal antibody production intermediate. In some aspects the virus-containing composition comprises a plasma-derived intermediate or plasma fractionation product. In some aspects the virus-containing composition comprises a vaccine bulk, viral seed train material, or viral propagation mixture.

[0316] In some aspects the virus-containing composition comprises an aqueous buffer selected from phosphate-buffered saline, Tris buffer, HEPES buffer, citrate buffer, or acetate buffer. In certain embodiments, the buffer comprises Tris at a concentration of about 10 mM to about 100 mM, and sodium chloride (NaCI) at a concentration of about 150 mM to about 2 M. In some embodiments, representative buffer formulations include 20 mM Tris and 500 mM NaCI or 80 mM Tris and 2M NaCI, optionally supplemented with the compound of this disclosure at a concentration of about 0.01% to about 5% (w / v), or about 0.5% to about 3% (w / v). Such buffer systems may be used in virus inactivation, cell lysis, or protein solubilization workflows and are compatible with the disclosed surfactant compounds.

[0317] In some aspects, provided are methods of inactivating a virus by contacting the virus with a composition as provided herein. In some aspects, the contacting is performed by mixing the compound with the virus-containing composition at a concentration from about 0.01% to about 5% (w / v) or about 0.5% to about 3% (w / v). In some aspects, the contacting is carried out for a period of about 10 minutes to about 2 hours, at a temperature ranging from about 20 °C to about 40 °C. In some aspects the virus-containing composition comprises a plasma-derived material selected from immunoglobulin preparations, albumin preparations, coagulation factor preparations, or antithrombin preparations.

[0318] In some aspects the virus or virus-containing composition is an intermediate or process stream from the production of a biopharmaceutical, vaccine, or plasma-derived product. In some aspects, contacting constitutes a surfactant-mediated virus inactivation step. In some aspects the compound of this disclosure is used in place of a non-ionic surfactant that produces environmentally persistent or endocrine-disrupting degradation products, such as alkylphenol ethoxylates, including octylphenol ethoxylates, nonylphenol ethoxylates, or commercial derivatives thereof. In some aspects the compound of this disclosure is formulated in an aqueous buffer comprising a salt and a stabilizer. In some aspects the stabilizer comprises glycerol.

[0319] In some other aspects the contacting is followed by a step of reducing the residual concentration of the compound to below 0.004% (w / v) or subjecting the composition to centrifugation to minimize cytopathic effect (i.e., visual or assay-based observations that indicate no substantial monolayer detachment, vacuolization, or cell death in treated host cells relative tobuffer or surfactant-only controls) in mammalian host cells used for viral infectivity assays. In some aspects the virus-containing composition is further treated to reduce the concentration of the compound to below 0.01% (w / v) prior to downstream purification. In some aspects, the method is used to reduce viral contamination in a biological or biopharmaceutical composition by rendering the virus non-infectious. In some aspects the compound of this disclosure is a biodegradable non-ionic surfactant. In some aspects, the composition does not cause visible cytopathic effect in mammalian host cells used for viral propagation

[0320] In some aspects, an exemplary protocol for virus inactivation, virus-containing samples are treated with 1% (w / v) of formulation containing Triton™ X-100 or a compound of this disclosure for 1 hour at 25 °C. Following treatment, samples are centrifuged at 20,000 x g for 2 hours at 4 °C, and the resulting pellets are resuspended in fresh culture medium. Infectivity is then assessed in indicator cells using a TCID50(median tissue culture infectious dose) assay, with viral titers quantified after a 7-day incubation. LRFs are calculated based on the difference in viral titer between treated and untreated control samples. In another format, inactivation efficacy is evaluated by treating virus samples with the disclosed compounds and infecting adherent cell lines under controlled conditions. After a defined incubation period, cells are fixed and stained with Dy Light™ 488-conjugated antibodies specific to the virus or its surface proteins, along with a nuclear stain (e.g., DRAQ5). Infectivity is assessed by measuring the presence or absence of fluorescence signal, while surf actant- related cytotoxicity is monitored by evaluating changes in cell morphology or adhesion. These assays collectively confirm that the compounds disclosed herein effectively inactivate viruses under biopharmaceutical process conditions.

[0321] Further, it is understood that the disclosed compounds are useful as surfactants and can be used in many applications where a nonionic surfactant is suggested or preferred. Applications include, but are not limited to, inactivating viruses, separating hydrophilic proteins from membranes, reducing surface tension, or decellularizing tissue.

[0322] In some aspects, compounds of this disclosure may be used in many applications relating to the manufacture of active pharmaceutical ingredients, biopharmaceuticals, plasma or cell-derived products and cell and gene therapies. For example, compounds of this disclosure may be used in processes involving viral inactivation, purification, contaminant removal, post-production cleaning, and quality control testing, thereby contributing to improved product quality, process efficiency, and regulatory compliance with established viral safety and product integrity standards. In another aspect, they may be used in the capture process for nonenveloped viruses, such as AAV, used in gene therapy. In vaccine development andproduction, compounds of this disclosure may be used in the virus splitting and inactivation steps.

[0323] In some aspects, compounds of this disclosure may be used in the production of recombinant proteins, antibodies, viral vectors, or other biological therapeutics, particularly in processes that employ human or animal cell lines. In these contexts, gentle surfactants are particularly desirable in order to effectively eliminate contaminants while maintaining the structural integrity, biological activity, and functional properties of the desired product. Compounds of this disclosure may be used in a variety of upstream and downstream purification processes in the production of biologic agents, including viruses, antibodies, and vaccines. Compounds of this disclosure may be used in such processes to remove liquid culture medium from cells, or in cell lysis applications to release intracellular components. They may also be used in various recovery, purification or isolation processes to remove or inactivate unwanted contaminants such as adventitious viruses or host cell proteins, lipids, and other impurities. Such processes include a variety of biochemical techniques including various types of chromatography, including but not limited to affinity chromatography, molecular sieve chromatography, cation exchange chromatography, hydrophobic interaction chromatography, or anion exchange chromatography, and filtration, including but not limited to molecular weight cutoff filtration, depth filtration using porous filter medium, or ultrafiltration.

[0324] Non-ionic surfactants are used extensively in bioprocessing and in the manufacture of active pharmaceutical ingredients, biopharmaceuticals, plasma or cell-derived products and cell and gene therapies. For example, non-ionic surfactants may be used in processes involving viral inactivation, purification, contaminant removal, post-production cleaning, and quality control testing, thereby contributing to improved product quality, process efficiency, and regulatory compliance with established viral safety and product integrity standards. In another aspect, they may be used in the capture process for non-enveloped viruses, such as AAV, used in gene therapy. In vaccine development and production, non-ionic surfactants are often used in the virus splitting and inactivation steps.

[0325] Non-ionic surfactants are commonly used in the production of recombinant proteins, antibodies, viral vectors, or other biological therapeutics, particularly in processes that employ human or animal cell lines. In these contexts, gentle surfactants are particularly desirable in order to effectively eliminate contaminants while maintaining the structural integrity, biological activity, and functional properties of the desired product. They may be used in a variety of upstream and downstream purification processes in the production of biologic agents, including viruses, antibodies, and vaccines. Non-ionic surfactants may be used in such processes toremove liquid culture medium from cells, or in cell lysis applications to release intracellular components. They may also be used in various recovery, purification or isolation processes to remove or inactivate unwanted contaminants such as adventitious viruses or host cell proteins, lipids, and other impurities. Such processes include a variety of biochemical techniques including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, hydrophobic interaction chromatography, or anion exchange chromatography) and filtration (e.g., molecular weight cut-off filtration, depth filtration using porous filter medium, or ultrafiltration).

[0326] Nonionic surfactants are also widely employed in endotoxin removal processes due to their ability to disrupt lipopolysaccharide (LPS) aggregation and modulate LPS-protein interactions without substantially denaturing sensitive biomolecules. These surfactants facilitate solubilization of hydrophobic endotoxin structures and reduce nonspecific binding of LPS to proteins, purification resins, and processing surfaces, thereby improving recovery and purity during biomanufacturing and research workflows. Incorporation of nonionic surfactants into buffer systems used for cell lysis, chromatographic purification, or final formulation enhances the efficiency of endotoxin removal and limits subsequent endotoxin rebinding. As a result, nonionic surfactants serve as critical auxiliary agents in achieving low-endotoxin preparations of recombinant proteins, vaccines, biologies, and analytical reagents. The skilled artisan will appreciate that the compound according to the present disclosure may be alternatives for other known nonionic surfactants in endotoxin removal processes.

[0327] In some aspects, compounds of this disclosure may be used in the manufacturing of compositions for use in viral inactivation, or in the manufacturing of compositions to reduce viral contamination in recombinant protein or monoclonal antibody production.

[0328] Nonionic surfactants are commonly employed in proteomic sample preparation owing to their ability to solubilize proteins, particularly hydrophobic and membrane-associated species, without contributing ionic interference that can hinder downstream analytical techniques. These surfactants facilitate efficient cell lysis, membrane disruption, and extraction of structurally diverse proteins while preserving native charge states and minimizing denaturation relative to ionic surfactants. In proteomic workflows, nonionic surfactants are used in multiple solutions to reduce protein aggregation and nonspecific adsorption, thereby improving recovery, reproducibility, and representation of low-abundance or difficult-to-extract proteins. The development of mass-spectrometry-compatible and acid-labile nonionic surfactants has further expanded their utility by enabling enhanced enzymatic digestion and straightforward removal priorto LC-MS analysis, reducing ion suppression and instrument contamination. Consequently, nonionic surfactants have become integral components of modern proteomic methodologies aimed at achieving comprehensive, high-sensitivity protein identification and quantification. One of skill in the art will readily understand the many uses that the compounds of this disclosure may have in proteomic workflows and associated reagents, including in various buffers and formulations used in cell lysis, membrane disruption, protein extraction, incubation solutions, forward probe solutions, and reverse probe solutions.

[0329] Lipid nanoparticles (LNPs) are an important class of delivery systems for nucleic acids, including messenger RNA (mRNA), small interfering RNA (siRNA), and genome editing components (e.g., ribonucleoproteins and the like). LNPs are typically formed by the rapid mixing of an organic lipid phase — comprising an ionizable lipid, helper lipids such as phospholipids and cholesterol, and PEG-lipids — with an aqueous phase containing a nucleic acid payload. Under these conditions, electrostatic interactions and hydrophobic assembly drive nanoparticle formation and encapsulation of the nucleic acid cargo.

[0330] Nonionic surfactants are widely used in biopharmaceutical formulations to reduce interfacial tension, prevent aggregation, and protect sensitive molecular structures from shear-induced degradation. The use of nonionic surfactants during one or more stages of LNP encapsulation, purification, or storage may enhance the robustness of LNP formation and encapsulation across diverse processing conditions. In particular, nonionic surfactants, such as Triton™ X-100, may be used to reduce particle aggregation during mixing, improve encapsulation efficiency, stabilize LNPs during downstream handling, and increase overall manufacturing yields provide significant advantages in the production of nucleic acid-containing lipid nanoparticles. There is a need for effective nonionic surfactants that can be used in LNP encapsulation but have a more favorable toxicology and environmental profile than other commercial nonionic surfactants.

[0331] Nonionic surfactants are also used, in certain concentrations, in LNP workflows in LNP encapsulation efficiency assays which test the efficiency of the encapsulation process. As demonstrated, compounds of this disclosure produced encapsulation-efficiency values comparable to those obtained with Triton™ X-100, providing comparable alternatives with more favorable environmental and toxicological profiles than current industry standards. In aspect, LNP encapsulation efficiency may be determined by determining the amount of free mRNA in a composition containing LNPs, contacting LNPs with a compound of this disclosure to release all mRNA, determining the amount of total mRNA, and calculating the percentage of mRNA encapsulated to arrive at an encapsulation efficiency %. In some aspects, compounds ofthis disclosure may be used in the contacting step in a concentration of about 0.001% -30% (v / v or w / v), or about 0.01% to about 20% (v / v or w / v). For example, the concentration of a compound of this disclosure might be about 0.01%, 0.05%, 0.1%%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.9%, 1%, 5%, 10%, 15%, or 20%, or any percentage between, One of skill in the art will readily understand how to optimize the concentration of a lysis buffer based on the chosen compound of this disclosure. As just one example, a compound of this disclosure might be used with a 1 x TE buffer in concentrations disclosed above, or in different concentrations, as a lysis buffer for use in an LNP encapsulation efficiency assay, to confirm the encapsulation workflow of a LNP process.

[0332] In some aspects, compounds of this disclosure may be used in LNP process workflows, including in encapsulation efficiency assays and at various stages during encapsulation, purification, or storage.Overview of Several Aspects

[0333] Disclosed herein is a compound having a structure according to Formula I,R4FY'Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

[0334] The compound may have a structure according to any one of Formulas IA-IFFormula IA Formula IBRI4R4R4^UR-°Formula IC Formula IDFormula IE Formula IF.

[0335] The R1and R2groups may be selected to provide a hydrophilic-lipophilic balance value ranging from 10 to 18.

[0336] The R1group comprises a heteroaliphatic group.

[0337] The heteroaliphatic is a linear heteroaliphatic group.

[0338] The linear heteroaliphatic group is a polyalkylene oxide.

[0339] The linear heteroaliphatic group is a PEG group having a mass average molecular weight ranging from 500 g / mol to 800 g / mol.

[0340] R1may comprise a cyclic heteroaliphatic group.

[0341] The cyclic heteroaliphatic group may comprise a sugar molecule.

[0342] R2may comprise an aliphatic group or an aryl group.

[0343] R2may comprise C225alkyl, phenyl, or naphthyl.

[0344] The C2 asalkyl group is linear, branched, cyclic, or a combination thereof.

[0345] p may be 1 or 2 and each R3independently may comprise an alkoxy group or an aliphatic group.

[0346] Each R4independently may comprise a Ci- alkyl group.

[0347] Each R4independently may comprise a methyl or ethyl.

[0348] n may be 1 and Y is oxygen or sulfur.

[0349] X may be oxygen or NH.

[0350] X may be oxygen.

[0351] The linker may not be present.

[0352] The linker may be present and is a Ci -asaliphatic group.

[0353] The linker may be present and has a structure according to the formula -C(=Z)-W-C(=Z)-X’-, wherein each Z independently is selected from O, S, or NR5; W is Ci-2salkyl, C1-2salkenyl, G1 -2salkynyl, ether, thioether, or amine; and X’ is O, S, or NR5.

[0354] The linker may be present and is -(CH2)q-, -C(=O)(CH2)qC(=O)-O-, or -C(=S)(CH2)qC(=S)-O-, -C(=O)N(H)(CH2)qN(H)C(=O)-O-, wherein each q independently is an integer ranging from 1 to 10.

[0355] m may be 1 and p may be 0.

[0356] The compound may have a structure according to any one of Formulas IG, IH, IJ, or IKFormula IJwherein the aliphatic group is linear, branched, or cyclic, or a combination thereof; TG, if present, is a terminating group that is an aliphatic group; and r is an integer selected from 2 to 20.

[0357] The aliphatic group may comprise a linear C2 2oalkyl group; a branched C22oalkyl group; a cyclic C3-2oalkyl group; or a combination of any such linear, branched, and / or cyclic groups.

[0358] The aliphatic group may comprise isopentyl, 2-methylpentyl, heptyl, 2,2-dimethylbutyl, pentyl, octyl, or nonyl.

[0359] The TG group may comprise a Ci- alkyl group.

[0360] The TG group may comprise a methyl.

[0361] In any or all of the above aspects, the compound may be selected from any one of the compounds 1 to 54 disclosed herein.

[0362] Also disclosed is a method, comprising exposing a sample to a compound disclosed herein.

[0363] The sample may comprise a biological sample.

[0364] The sample may comprise a cell. The cell may be solubilized and / or permeabilized.

[0365] Also disclosed is a method of lysing a cell, comprising contacting the cell with an amount of a compound disclosed herein sufficient to facilitate cell lysis.

[0366] Also disclosed is a composition, comprising a compound disclosed herein.

[0367] Also disclosed is a kit, comprising a compound disclosed herein and a container.

[0368] The present disclosure also relates to scientific analysis systems, as well as related computer-implemented methods, computing devices, and computer-readable media, that utilize data generated by processes that involve the novel compounds disclosed herein.

[0369] For example, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may utilize data generated as part of imaging applications involving any of the novel compounds disclosed herein, such as immunohistochemistry, FISH, cell tracing, receptor labeling, and cytochemistry. In another example, these systems, computer-implemented methods, computing devices, and computer readable media may utilize data generated by use of the novel compounds disclosed herein for probing biological structure, function, and interactions.

[0370] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to detect at least one biomolecule based on data indicative of the combination of the at least one biomolecule with a composition comprising at least one excipient and a compound of Formula (I) this disclosure in an effective concentration to label the at least one biomolecule under conditions sufficient for binding the compound to the biomolecule.

[0371] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may utilize data generated by an assayselected from fluorescence microscopy, flow cytometry, in vivo imaging, immunoassay, hybridization, chromatographic assay, electrophoretic assay, microwell plate based assay, FRET system, BRET system, high throughput screening, or microarray. In another example, the data may be generated by in vivo imaging comprising providing the biomolecule-bound compound to at least one of a biological sample, tissue, or organism, and detecting the biomolecule within the at least one of a biological sample, tissue, or organism.

[0372] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to use data to detect or quantify biological material using fluorescent dyes including the novel compounds disclosed herein. For example, the data may be generated by optical techniques, including fluorescence optical, qualitative and / or quantitative determination methods to diagnose properties of cells (molecular imaging), in biosensors (point of care measurements), for investigation of the genome, or in miniaturizing technologies. In another example, the data may be generated by microscopy, super-resolution imaging, cytometry, cell sorting, PCS, uHTS, me-FISH, FRET-systems, BRET systems, or microarrays (DNA- and protein-chips). In some embodiments, the systems, computer implemented methods, computing devices, and computer-readable media disclosed herein may be configured to use data indicative of fluorescence emission to perform any detection or quantitation techniques involving the novel compounds disclosed herein.

[0373] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to use data to detect or quantify antigens using the novel compounds disclosed herein. For example, the data may be generated by immunofluorescence, immunohistochemistry, flow cytometry (e.g., FACS), Western blotting, fluorescence ELISA, and any other approach where fluorescently labeled antibodies can be used.

[0374] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to use data (e.g., fluorescent signals) to assess cell viability generated by techniques utilizing the novel compounds disclosed herein. For example, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may use data to quantifying the amount of live and / or dead cells, e.g., by determining whether the degree of fluorescence exceeds a predetermined threshold (indicating nonviability), or by grouping results into live anddead populations (where dead cell populations show a greater extent of staining given their compromised membrane integrity).

[0375] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to exclude dead cells from data generated by techniques utilizing the novel compounds disclosed herein, allowing cleaner separation and identification of cell populations.

[0376] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to collect and analyze fluorescence data generated by techniques utilizing the novelcompounds disclosed herein. In some embodiments, the data may be generated by a plate reader, fluorescence microscope, or flow cytometer. In certain embodiments, the fluorescence detection can be achieved using an acoustic focusing flow cytometer, such as the Attune ™ NxTTMFlow Cytometer from Thermo Fisher Scientific. In other embodiments, fluorescence detection can be achieved using a cell sorting flow cytometry instrument, such as the Invitrogen Bigfoot Spectral Cell Sorter from Thermo Fisher Scientific.

[0377] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to spectrally distinguish multiple fluorescent molecules, as disclosed herein.

[0378] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to collect or analyze data generated by techniques involving the novel compounds disclosed herein and prolonged heat exposure and / or extended irradiation, such as PCR and fluorescence imaging.

[0379] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to collect or analyze data generated by techniques involving the novel compounds disclosed herein, such as multiplex flow cytometry experiments where bright and stable fluorescent dyes in this region of the spectrum are needed, multicolor panels in both conventional and spectral flow cytometry applications, or multi-color imaging experiments where bright and stable fluorescent dyes in this region of the spectrum are needed.

[0380] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to collect or analyzedata generated by techniques involving the novel compounds disclosed herein, such as labeling or detecting nucleic acids in a wide variety of samples, such as in aqueous solutions, sequencing or amplification reactions such as PCR, cellular samples (e.g., for FISH or general nuclear or chromosome staining) and electrophoretic gels.

[0381] In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to collect or analyze data generated by techniques involving the novel compounds disclosed herein, with examples of such data including characteristics of a compound-nucleic acid complex, including the presence, location, intensity, excitation and emission spectra, fluorescence polarization, fluorescence lifetime, and other physical properties of the fluorescent signal. In some embodiments, the systems, computer-implemented methods, computing devices, and computer-readable media disclosed herein may be configured to collect or analyze data generated by techniques involving the novel compounds disclosed herein to detect, differentiate, sort, quantitate, and / or analyze aspects or portions of the sample.

[0382] Any of the computational methods discussed herein may include any known data collection, storage, analysis, and decision-making techniques. For example, any suitable ones of the computational methods discussed herein may include machine-learning or other analytical techniques to process and understand the data generated by scientific experiments and other processes involving the novel compounds disclosed herein. Such techniques may include supervised learning (i.e., training a machine learning model using labeled data to predict or classify new observations), unsupervised learning (i.e., identifying patterns and structures within data without pre-existing labels using techniques such as clustering), dimensionality reduction (i.e., techniques for simplifying or filtering a complex data set, such as principal component analysis (PCA)), or deep learning (i.e., utilizing neural network computational methods with multiple layers to extract intricate patterns and representations from data), statistical analysis (e.g., hypothesis testing, regression analysis, analysis of variance (ANOVA), and correlation analysis.

[0383] In some embodiments, any suitable ones of the computational methods disclosed herein may include analyzing data, generated by imaging (e.g., fluorescence microscopy), using machine learning, as known in the art. Examples of such embodiments may include using machine-learning to perform object detection and segmentation in imaging data (e.g., to identify cells or subcellular structures), image classification (e.g., to classify differenttypes of structures or identify specific cellular events based on patterns in fluorescence images), or performing image quality and denoising.

[0384] In some embodiments, any suitable ones of the computational methods disclosed herein may include analyzing data, generated by flow cytometry, using machinelearning, as known in the art. Examples of such embodiments may include using machinelearning to perform cell population identification and classification (e.g., by identifying and classifying different cell types) or to perform correction for artifacts (e.g., spectral overlap or fluorescence spillover).

[0385] Also, provided herein are scientific instruments that can detect a compound, as disclosed herein. In certain embodiments, the compound is bound to a biomolecule or biological material as disclosed in FIGS 1-12. In certain embodiments, the scientific instrument can be used to generate a detectable response from the compound and can further include a computational device fix analysis of the detectable response. Detection of the detectable response by the computational device can further include performing a machine-learning analysis technique on a data set of detectable responses generated by the instrument. The machine-learning technique can use a computer process to generate a machine learning model, and the machine learning model based on the model data and a prediction process can be trained to predict and / or classify new observations.

[0386] Thus, in one aspect a method is provided that includes determining an attribute, by a computing device, as a function of a machine-learning process, wherein data generated from one or more dyes, as disclosed herein, and collected with an analyzer electronically connected to the computing device is inputted into the machine-learning process. An analyzer (e.g., a scientific instrument; that is electronically connected to a computing device is used to generate data from a compound, as disclosed herein. The data serves as input for a machinelearning process that is conducted by the computing device, where the attribute canbe determined by the computing device as a function of the machine learning process, In some embodiments, the output of the machine learning process provides information about the attribute. It is contemplated that the described method can be used by any type of analyzer, including, but not limited to, a fluorescence microscope, flow cytometer, cell sorter, mass spectrometer, mass cytometer, imaging cytometer, fluorescence resonance energy transfer (FRET) system, bioluminescence resonance energy transfer (BRET) system, and an electron microscope. In some embodiments, the analyzer is configured to collect data for an assayselected from the group consisting of an in vivo imaging assay, an immunoassay, a hybridization assay, a chromatographic assay, an electrophoretic assay, a microwell platebased assay, a high throughput screening assay, and a microarray-based assay; or to collect data gene.

[0387] In another aspect, a machine-learning process can be included in the method for configuring the analyzer prior to collection of data generated from contacted samples. The analyzer can be configured with a set of configuration settings, where the configuration settings can be determined based on the type of sample to be interrogated or the desired type of analysis or experiment. The configuration settings can be adjusted manually or can be automated. In certain embodiments, the configuration settings can be determined using a machine learning process that is distinct from machine learning process used to determine an attribute based on the data generated from a sample contacted with permeabilization buffer. An exemplary method can include processing data from an analyzer using a machine learning process to adjust or predict configuration issues to adjust configuration settings associated with the analyzer. The method can configure settings based on the type of sample to be interrogated or to aid in selection of an appropriate experiment by_the researcher. Representative examples of configuration settings include, but are not limited to, voltage (e.g., plate voltage, photomultiplier tube voltage, or electrical charging ring voltage, area selection, flow rate, filter position, gates, alignment and calibration settings, wavelength or laser selection, and the like. In a specific example, the analyzer can be a flow cytometer or cell counting system, and the configuration settings can include, e.g., voltage (e.g., plate voltage, photomultiplier tube voltage, or electrical charging ring voltage) or an area corresponding to gating of cells.

[0388] The attribute that can be determined using methods disclosed herein can be any property or characteristic relating to the sample that is being interrogated by the analyzer, such as a structure, a function, or an interaction, For example, the attribute can be a physical or chemical characteristic of a structure, such as a molecule, particle, or a biological material (e, g., a cell or tissue). The attribute of the structure can be, for example, size, shape, complexity, concentration, chemical composition, and the like. In certain embodiments, the attribute can be an interaction, such as the interaction between the compound (or a substance to which the compound is bound) and a structure. For example, the attribute can be information about the type and / or number of structures (e.g., cells or molecules) to which the compound is bound. The interaction can be between the compound (or a substance to which the compound is bound; and another molecule). By way of a specific example, a cell contacted with a permeabilizationbuffer, as disclosed herein, can be bound to a protein (e.g., an antibody) that can recognize a protein on the surface of or within the interior of a biological cell. Upon integration by an analyzer, such as a fluorescence microscope or flow cytometer, the analyzer detects an emission signal from the bound compound. The data can be input into a computing device that is electronically connected to the analyzer, The computing device can conduct a machinelearning process on the emission data from the compound, hereby the machine learning process determines an attribute, such as the identity or concentration of a cell population, In other embodiments, the attribute can be a function, such as cellular function, including without limitation, protein expression patterns and gene expression, live and dead cell distinction, or cell cycle stage.

[0389] In certain embodiments, the attribute is a spectral property, a measure of spectral overlap or an artifact in the data. In certain embodiments, the machine learning process corrects artifacts in the data, removes noise, or normalize the data, such that the data is in a suitable format for subsequent analysis. Thus, certain methods include processing the data using the computing device to remove noise, correct for artifacts, and / or to normalize the data, such that the data is in a suitable format for subsequent analysis. Certain methods further include one or more of the following steps; extracting one or more features from the processed data to capture detectable responses of interest; training the machine learning process from the extracted features, such that the process learns patterns or relationships between the extracted features; validating the trained machine learning process to assess the performance and ability of the process to accurately classify or predict patterns or relationships based on the detectable responses; or applying the trained process to predict or classify detectable responses by making predictions based on the trained patterns or relationships. In certain embodiments, methods disclosed herein can further include training, by the computing device, the machinelearning process, using training data correlating the data from the one or more bound compounds to the attribute.

[0390] In yet another aspect, provided herein is one or more non-transitory computer-readable media having instructions thereon that, when executed by one or more processing devices of one or more computing devices, cause the one or more computing devices to perform the method disclosed herein. Also provided herein is a computing device configured to perform the method described herein is provided.

[0391] In yet another aspect an apparatus is provided that includes a computing device, wherein the computing device comprises: at least one processor comprising memory, wherein the memory includes instructions to: determine an attribute as a function of a machine-learning process, wherein data generated from one or more contacted cells, and collected from an analyzer electronically connected to the computing device is inputted into the machine learning process.

[0392] In any of the methods involving a machine-learning process or apparatus for performing a machine-learning process, one or more contacted cell compounds, as disclosed herein, can be bound to a biomolecule, such as, for example, a protein, polypeptide, antibody, enzyme, nucleic acid, nucleoside triphosphate,, oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid, carbohydrate, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, fluorescent proteins, or a combination of biomolecules. In any of the methods involving a machine-learning process, a dye, for example, can emit light in a region of the electromagnetic spectrum upon excitation at an appropriate wavelength of light. In some cases, the dye can have an excitation profile in the far- red region of the electromagnetic spectrum. In other cases, the dye has an excitation profile in the near-IR region of the electromagnetic spectrum. The dye or other detectable label may be as described elsewhere herein.

[0393] Methods described herein can be implemented with any number of scientific instruments adapted for detecting a response from a compound, as disclosed herein. By way of illustration, one representative method implements a flow cytometer to detect one or more optical signals generated by a sample. The sample can include one or more compounds, as disclosed herein. In certain embodiments, the compound is bound to or incorporated into a cell. In certain embodiments, the method can be used to analyze a sample that includes a plurality of cells, where at least one cell in the sample is bound to the compound. A computational device can be associated with the flow cytometer to analyze data generated by the instrument. Machine learning techniques can be particularly useful for processing large and complex datasets produced from samples that include a multitude of different detectable responses. For example, a sample can include a compound, as disclosed herein, and can further include one or more other types of detectable labels to provide a sample that produces multiple types of detectable responses when analyzed using a scientific instrument. In certain embodiments, the collected data includes a series of detectable responses from the one ormore detectable labels (e.g., dyes), For example, the detectable response canbe fluorescence emission and / or light scattering.

[0394] When methods are used to process data generated by a flow cytometer, such methods can leverage machine-learning for various purposes. Machine learning processes can be used to generate training sets that leverage a host of data produced by the flow cytometer. In one example, machine learning is used to remove noise, correct artifacts, and normalize data. In another example, machine learning can aid in clustering and cell population identification. Machine learning processes can be applied to identify distinct cell populations within a flow cytometry data set by grouping cells based on similarity in marker expression patterns, thereby allowing for identification or rare or abnormal cell populations. In another example, machine learning can be used to help visualize data more effectively by reducing dimensionality resulting from high dimensional datasets that contain many markers. In yet another example, machine learning can be used for classification and prediction, e.g., to classify cells in categories based on marker expression patterns that distinguish between healthy and diseased cells.

[0395] The compounds of the disclosure, together with the experimental and / or computational data provided herein characterizing their physicochemical, biological, and formulation-related properties, can be utilized as a training set and / or reference dataset for one or more artificial intelligence (Al) or machine-learning (ML) models configured to recognize structure-property relationships and to identify, design, or prioritize additional compounds exhibiting similar or improved advantageous properties (for example, enhanced solubility, stability, activity, selectivity, permeability, or compatibility with a given assay or workflow). Such Al or ML models may be used, without limitation, to perform virtual screening, de novo compound design, lead optimization, or in silico prediction of performance in relevant applicatio...

Claims

We claim:

1. A cell permeabilization buffer, comprising:a surfactant according to Formula I;Rz0R Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic,or heteroaliphatic; anda polar protic solvent, wherein the concentration of the surfactant is between 0.005% to 5% (v / v), wherein the compound is included in an amount sufficient to permeabilize a cell.

2. A cell permeabilization buffer, comprising:a surfactant according to Formulas I A, IB, IC, ID, IE, or IF:Formula IA Formula IBFormula IC Formula IDR*o. <Formula IE Formula IF.; anda polar protic solvent, wherein the concentration of the surfactant is between 0.005% to 5% (v / v), wherein the compound is included in an amount sufficient to permeabilize a cell.

3. A cell permeabilization buffer, comprising:a surfactant according to Formulas IG, I H, I J, or IK:LH or TGFormula IH Formula IGFormula IJFormula IKwherein the aliphatic group is linear, branched, or cyclic, or a combination thereof; TG, if present, is a terminating group that is an aliphatic group; and r is an integer selected from 2 to 20; anda polar protic solvent, wherein the concentration of the surfactant is between 0.005% to 5% (v / v), wherein the compound is included in an amount sufficient to permeabilize a cell.

4. A cell permeabilization buffer, comprising:a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1 - 54:CM; anda polar protic solvent, wherein the concentration of the surfactant is between 0.005% to 5% (v / v), wherein the compound is included in an amount sufficient to permeabilize a cell.

5. The cell permeabilization buffer of any of claims 1 -4, further comprising one or more of a salt, a buffering agent, a blocking agent, a fixative quencher, and an enzyme.

6. The cell permeabilization buffer of any of the preceding claims, wherein the fixative quencher is selected from the group consisting of glycine, ammonium chloride, Tris, ethanolamine, lysine, sodium borohydride, serum, bovine serum albumin, or any combination thereof.

7. The cell permeabilization buffer of claim 5 or 6, wherein the blocking agent is selected from the group consisting of albumin, casein, gelatin, and BSA.

8. The cell permeabilization buffer of any of claims 5-7, wherein the enzyme is a protease or lysis enzyme.

9. The cell permeabilization buffer of any of claims 5-7, wherein the enzyme is selected from the group consisting of trypsin, proteinase K, pepsin, pronase,Papain, lysozyme, zymolyase, achromopeptidase,lysostaphin, labiase, kitalase, lyticase, glucanase, collagenase, dispase, or any combination thereof.

10. A method for permeabilizing a cell, comprisingcontacting the cell with a cell permeabilization buffer according to any one of claims 1-9, wherein the permeabilization buffer comprises an amount of a surfactant effective to facilitate permeabilization of the cell.

11. The method of claim 10, wherein the cell is selected from the group consisting of a eukaryotic cell, a plant cell, a fungal cell, and a bacterial cell.

12. The method of claim 10 or 11, wherein the cell is selected from the group consisting of blood cells, immune cells, cultured cells, or primary cells.

13. A method of detecting one or more analytes in a biological sample comprising a cell, comprising:(i) contacting the sample with a cell permeabilization buffer according to any one of claims 1-9 for a period of time to permeabilize the cell;(ii) optionally contacting the sample with a fixation buffer before, after, or simultaneously with the cell permeabilization buffer;(iii) incubating the sample with an analyte binding agent for a time interval adequate to allow entry of the analyte binding agent into the cell;(iv) optionally washing the sample to remove any unbound analyte binding agent; and (v) analyzing the sample to detect the analyte binding agent.

14. The method of claim 13, wherein the sample is contacted with a plurality of analyte binding agents, wherein each of the plurality of antibody binding agents specifically binds to a different analyte.

15. The method of claim 13 or 14, wherein the sample is a tissue sample, a cell culture, or an environmental sample.

16. The method of any one of claims 13-15, wherein analyzing the sample comprises direct detection of the analyte binding agent.

17. The method of any one of claims 13-16, wherein analyzingthe sample comprises indirect detection of the analyte binding agent.

18. The method of claim 17, wherein the indirect detection of the analyte binding agent comprises: contacting the sample with a secondary analyte binding agent that specifically binds to the analyte binding agent, and detecting the secondary binding agent.

19. The method of claim 17, wherein the indirect detection of the analyte binding agent comprises contacting the sample with a reagent that produces a detectable signal upon binding to the analyte binding agent.

20. The method of any one of claims 13-19, wherein the analyte binding agent specifically binds to an analyte selected from the group consisting of a protein, a nucleic acid, a carbohydrate, a lipid, a metabolite, or any combination thereof.

21. The method of claim 20, wherein the analyte binding agent is an antibody, antibody fragment, aptamer, or oligonucleotide.

22. The method of any one of claims 13-21, wherein the analyte binding agent comprises a detectable label.

23. The method of any one of claims 10-22, comprising performing steps (iii)-(v) on the sample between 1 and 20 times.

24. The method of any one of claims 10-22, wherein the sample is contacted with between 1 and 100 different analyte binding agents, wherein each different analyte binding agents specifically binds to a different analyte.

25. The method of any one of claims 10-24 wherein analyzing thesample comprises contacting the sample with a polymerase and / or transcriptase under conditions to permit nucleic acid polymerization and / or amplification.

26. The method of any one of claims 10-25, wherein the surfactant is administered in a concentration of about 0.005%, 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5% or any value between.

27. A kit, comprisinga cell permeabilization buffer according to any one of claims 1-9; and one or more reagents selected from the group consisting of: a fixation buffer, a mountant, a wash buffer, an analyte binding agent, a gel, a matrix, a membrane, or any combination thereof.

28. Use of a surfactant according to any one of claims 1 -9 to permeabilize a cell.

29. The use of claim 28, wherein the permeabilization buffer permeabilizes the cell membrane, the nuclear membrane, or both.

30. A loading buffer for gel electrophoresis to separate proteins or nucleicacids, comprising a surfactant according to Formula I:R* M6... JM,M > u.p*Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic, a tracking dye, and optionally glycerol and / or a reducing agent.

31. Use of a surfactant according to wherein the surfactant is a compound having a structure according to Formula I:JFormula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic,in the manufacture or formulation of a tracking dye for gel electrophoresis.

32. A blocking buffer, comprisinga surfactant according to Formula I:R4Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic,a tracking dye, and optionally glycerol and / or a reducing agent;a blocking agent; anda buffering agent.

33. The blocking buffer of claim 32, wherein the blocking agent is selected from the group consisting of non-fat dry milk, albumin, BSA, casein, and gelatin.

34. A kit, comprising, a blocking buffer according to claim 32 or 33, andoptionally one or more of a wash buffer, a gel, a transfer membrane, a lateral flow strip, and one or more analyte binding agents,wherein the concentration of surfactant is effective to inhibit non-specific interactions between polypeptides while maintaining specific binding between analytes and analyte binding agents.

35. Use of a surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic,in the manufacture or formulation of a blocking buffer.

36. A method of reducing non-specific binding of an antibody or antibody fragment to a membrane or a matrix in an assay, comprising;providing a membrane or matrix, andcontacting the membrane or matrix with a blocking buffer according to claim 32 or 33, thereby reducing non-specific binding of the antibody or antibody fragment to the membrane or matrix.

37. The method of claim 36, wherein the membrane or matrix is bound by or embedded with an analyte, prior to the contacting step.

38. The method of claim 37, wherein the analyte is a polypeptide or a nucleic acid.

39. The method of claim 36, wherein the assay is a Western Blot, an ELISA assay, a dot blot, a Northern Blot, a Southern Blot, or a lateral flow assay.

40. The method of claim 36, further comprising contacting the analyte-bound or analyte- embedded membrane or analyte bound or analyte-embedded matrix with an analyte binding agent.

41. The method of any one of claims 36-40, wherein the membrane or matrix is selected from a nitrocellulose membrane, a PVDF membrane, and an affinity column matrix.

42. The method of any of claims 36-41, further comprising washing the analyte-bound or analyte-embedded membrane, or the analyte-bound or analyte-embedded matrix with a wash buffer.

43. A mountant, comprising:a surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic,or heteroaliphatic; anda base medium.

44. The mountant of claim 43, wherein the base medium comprises one or more of an antifade or fluorophore stabilizer, a refractive index modifier, a preservative, a hardening agent, a wetting agent, or a clearing agent.

45. Use of a surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic,in the manufacture of a mountant.

46. A composition, comprising:a bead or microcarrier conjugated to an analyte-binding agent; anda surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X' is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

47. A kit comprising:a capture antibody reagent, wherein the capture antibody reagent comprises a bead or microcarrier conjugated to a first analyte binding agent, wherein the analyte binding agent is an antibody or fragment thereof or a non-antibody capture agent that specifically binds a first epitope on a target analyte;a detection antibody reagent, wherein the detection antibody reagent comprises a detectable label, wherein the second analyte binding agent specifically binds a second epitope on the target analyte; andoptionally a lysis buffer;optionally a wash buffer;wherein one or more of the capture antibody reagent, the detection antibody reagent, the lysis buffer, or the wash buffer comprises a surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

48. The kit of claim 47, wherein the capture antibody reagent comprises a bead or microcarrier that is internally labeled with a dye.

49. The kit of claims 47 or 48, comprising a plurality of capture binding reagent / detection antibody reagent pairs, wherein each pair specifically binds to a first and second epitope on a unique target analyte.

50. The kit of any one of claims 47-49, comprising 2 to 100 capture binding reagent / detection antibody reagent pairs.

51. The kit of any one of claims 47-50, wherein the detectable label is selected from the group consisting of a radiolabel, an organic fluorophore, a fluorescent protein, a quantum dot, a chromophore, an enzymatic label, a chemiluminescent label, a bioluminescent label, a metalbased label, an oligonucleotide-based label, an affinity tag, or any combination thereof.

52. A sample application pad for use in a lateral flow assay device, the sample application pad comprising:(a) a porous substrate configured to receive a liquid biological sample and to regulate the flow of the sample into a downstream conjugation zone;1. a treatment composition disposed on or within the porous substrate, the treatment composition comprising:i. a nonionic surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphaticor heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic,present in an amount effective to enhance sample wetting and promote release of a target analyte from the biological sample; andii. optionally one or more of a buffer, a blocking agent, and a stabilizer;wherein the sample application pad is configured such that, upon application of the biological sample, the treated porous substrate improves capillary flow uniformity of the sample, and delivers the sample to the downstream region of the lateral flow assay device.

53. A lateral flow assay device for detecting a target analyte in a biological sample, the device comprising:(a) a sample application pad according to claim 52;(b) a conjugate pad in fluid communication with the samplepad, wherein the conjugate pad contains a dried detection reagent comprising an analyte binding agent that comprises a detectable label and that specifically binds a first epitope on the target analyte forming a detection reagent-target analyte complex, when the target analyte is present, wherein the label selected from the group consisting of colloidal gold, latex particles, enzymes, and fluorescent dyes;(c) a porous membrane comprising:i. a test zone having an immobilized capture binding member configured to specifically bind the target analyte-detection reagent complex; andii. a control zone having an immobilized control binding member configured to excess detection reagent that is not bound to the target analyte;d. an absorbent pad positioned downstream of the porous membrane and configured to draw the sample fluid through the device via capillary action; ande. a housing that supports the sample pad, conjugate pad, membrane, and absorbent pad in a linear flow path,wherein, upon application of the biological sample to the sample pad, the sample flows laterally through the device, rehydrates the detection reagent, and wherein the detection reagent-target analyte complex produces a visually or instrumentally detectable signal in the test zone indicative of the presence of the target analyte in the biological sample.

54. A running buffer, comprisinga surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic, in an amount effective to improve capillary flow of the sample through a lateral flow strip.

55. Use of a surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic, in the manufacture of a lateral flow assay device or a lateral flow strip.

56. A kit comprising:an analyte capture reagent / analyte detection reagent pair,wherein the analyte capture agent comprises an analyte binding agent afirst oligonucleotide probe, wherein the analyte binding agent specifically binds to a first epitope on a target analyte;an analyte detection agent comprising analyte binding agent and a second oligonucleotide probe, wherein the analyte binding agent specifically binds to a second epitope on the target analyte;optionally, a splint oligonucleotide probe that is complementary to at least a portion of the first oligo probe and at least a portion of the second oligonucleotide probe;optionally, a lysis buffer;optionally, a DNA ligase;optionally, a DNA polymerase;optionally, dNTPs; andoptionally, an amplification buffer;wherein one or more of the analyte capture reagent, analyte detection reagent, or lysis buffer comprises a surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

57. A method comprising:contacting a biological particle with a permeabilization buffer comprising a surfactant according to any one of claims 1-9 in an amount effective to facilitate permeabilization of the biological particle;analyzing the biological particle with an analyzer to detect one or more signals indicative of permeabilization or target labeling; anddetermining an attribute, by a computing device, as a function of a machine-learning process, wherein data generated from the biological particle and collected with the analyzer electronically connected to the computing device is input into the machine-learning process, and wherein the attribute is associated with performance of the surfactant or the permeabilization buffer.

58. The method of claim 57, wherein the attribute comprises a classification or quantitative measure of one or more of: permeabilization efficiency, intracellular target accessibility, surfactant concentration suitability, lot-to-lot surfactant consistency, or stability of the permeabilization buffer.

59. The method of any of claims 57 or 58, wherein the collected data comprise a series of detectable responses from biological particles contacted with a plurality of permeabilization buffers comprising different concentrations of the surfactant of claims 1-9, and wherein the machine-learning process is configured to recommend an optimal surfactant concentration or incubation condition.

60. The method of any of claims 57-59, wherein the detectableresponses comprise fluorescence emission, light scattering, or a combination thereof from oneor more detectable labels introduced after permeabilization with the permeabilization buffer comprising the surfactant according to claims 1-9.

61. The method of any of claims 57-60, further comprising training the machine-learning process as a function of extracted features that characterize the effect of the surfactant or the permeabilization buffer on the biological particles, the extracted features comprising one or more of: signal intensity, signal-to-background ratio, cell morphology, cell viability, fraction of labeled cells, or distribution of intracellular marker intensity.

62. The method of any of claims 57-61, wherein the analyzer is configured to collect data from an assay that utilizes the permeabilization buffer comprising the surfactant according to claims 1-9, the assay selected from the group consisting of an intracellular immunoassay, an intracellular cytokine staining assay, an imaging assay of intracellular targets, a bead-based multiplex protein assay performed on a lysate generated using the surfactant, a Western blot assay using a lysate generated using the surfactant, or a gene expression assay using RNA extracted from cells permeabilized or lysed with the surfactant.

63. The method of any of claims 57-62, further comprising configuring one or more parameters of the permeabilization procedure using a second machine-learning process, wherein the second machine-learning process is trained to adjust at least one of surfactant concentration, permeabilization time, temperature, buffer composition, or wash conditions to achieve a target permeabilization performance metric.

64. A system comprising:an analyzer configured to detect one or more signals from biological particles that have been contacted with a permeabilization buffer comprising a surfactant according to claims 1-9; and a computing device electronically connected to the analyzer, the computingdevice comprising at least one processor and memory storing instructions that, when executed by the at least one processor, cause the computing device to:receive data generated by the analyzer from the biological particles;apply a machine-learning process to determine at least one attribute associated with performance of the surfactant or the permeabilization buffer as in any of claims 54-60; and optionally output one or more recommended surfactant- or permeabilization-related parameters for use in subsequent assays.

65. A composition comprising a surfactant according to wherein the surfactant is a compound having a structure according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic, in an amount effective to stabilize the polypeptide, a buffering agent, and a polypeptide.

66. A composition according to claim 65 where the composition is a polypeptide storage buffer, a polypeptide reaction buffer, an immunoassay reaction buffer, an enzymaticreaction buffer, Master Mix, a running buffer, or a chromatography elution buffer.

67. The composition of claims 65 or 66, wherein the composition consists essentially of a buffering agent, a polypeptide and, optionally, one or more of a salt, a reducing agent, a chelating agent, a cryoprectant, a stabilizer or a carrier.

68. The composition of any of claims 65-67, wherein the polypeptide is an enzyme, a growth factor, a cytokine, a matrix protein, an antibody or fragment thereof, or an antigen.

69. The composition of claim 68, wherein the enzyme is selected from the group consisting of: a nuclease, a polymerase, a reverse transcriptase, a ligase, a glycosylase, a nuclease inhibitor, an alkaline phosphatase, an isomerase, a transferase, an oxidoreductase, and a lyase.

70. The composition of any of claims 67-69, wherein the salt comprises monovalent and / or divalent cation salts.

71. The composition of any of claims 65-70, wherein the composition further comprises EDTA, DTT, BSA and / or glycerol.

72. The composition of any of claims 65-71, wherein the surfactant is present in the composition at a concentration from about 0.0001 %-10% (v / v).

73. A method for stabilizing one or more polypeptides, the method comprising: combining said one or more polypeptides and a buffering agent with a compound having a structure according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic, thereby producing a composition.

74. The method of claim 73, wherein the polypeptide is an enzyme.

75. The method of claim 74, wherein the enzyme is the enzyme is selected from the group consisting of: a nuclease, a polymerase, a reverse transcriptase, a ligase, a glycosylase, a nuclease inhibitor, an alkaline phosphatase, an isomerase, a transferase, an oxidoreductase, and a lyase.

76. The method of any of claims 73-75, wherein the method further comprises lyophilizing or air-drying the composition.

77. A kit comprising a composition according to any of claims 65-72.

78. A method of determining LNP encapsulation efficiency, by determining the amount of free mRNA in a composition containing LNPs, contacting LNPs with a surfactant according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic, in an amount sufficient to release all mRNA, determining the amount of total mRNA, and calculating the percentage of mRNA encapsulated.

79. A kit for determining LNP encapsulation efficiency, comprising lysis buffer with a surfactant, wherein the surfactant is a compound according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

80. The buffer of any one of claims 1-5, 32, or 66-68, wherein the buffering agent is selected from the group consisting of: citrate buffers (saline-sodium citrate (SSC)),phosphate buffers (phosphate buffered saline (PBS)), tris (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-1,3-diol)-based buffers, TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid)-based buffers, HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid)-based buffers, Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid)-based buffers, tricine (N-[tris(hydroxymethyl)methyl]glycine)-based buffers, TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)-based buffers, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)-based buffers, MES (2-(N-morpholino)ethanesulfonic acid)-based buffers, PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid))-based buffers, Cacodylate (dimethylarsenic acid)-based buffers, or any combination thereof.

81. The methods of claim 22-26 or 60, or the kit of claim 47, wherein the detectable label is selected from the group consisting of a radiolabel, an organic fluorophore, a fluorescent protein, a quantum dot, a chromophore, an enzymatic label, a chemiluminescent label, a bioluminescentlabel, a metal-based label, an oligonucleotide-based label, an affinity tag, a hapten, a detectable substrate, colorimetric protein or peptide detection dye, or any combination thereof.

82. The method of claim 81, wherein the fluorophore is chosen from a phycoerythrin, a xanthene, a fluorescein, a rhodamine, a rhodol, a roseamine, a carbopyranone, an indole, an indacene, a borapolyazaindacene, a furan, a benzofuran, a cyanine, a benzocyanine, a benzopyrilium, a pyrene, a coumarin, a carbostyryl, a styryl, a squarine, a resorufin, an anthraquinone, an acridine, a benzophenoxazine, a cyanine-based tandem dye, phycoerythrin-dye conjugates, allophycocyanin, allophycocyanin-dye conjugates, a nanocrystal, a Pdot, a fluorescent conjugated polymer, or an oligonucleotide-based fluorescent dye.

83. The method of claim 81, wherein the chromophore is chosen from 3,3'-diaminobenzidine, 4-chloro-2-methylbenzenediazonium (Fast Red), 3,3'-dimethoxybiphenyl-4,4'-di(diazonium) or zinc chloride (Fast Blue).

84. The method of claim 81, wherein the hapten is chosen from fluorescein, biotin, nitroaryls, dinitrophenol (DNP), digoxigenin, oxazole, pyrazole, thiazole, benzofuran, urea, thiourea, rotenoid, coumarin, or cyclolignan.

85. The method of claim 81, wherein the enzymatic label is chosen from peroxidase, a phosphatase, a glycosidase, or an oxidase.

86. The method of claim 81, wherein the detectable substrate comprises a substrate for a peroxidase, a substrate for horseradish peroxidase, a tyramide, or a tyramide-like molecule.

87. The method of claim 81, wherein the colorimetric protein or peptide detection dye comprises bicinchoninic acid (BCA), bathocuprione, bathocuprionedisulphonic acid, tartrate, copper sulphate, acetonitrile, salts thereof, derivatives thereof, and combinations thereof.

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