WO2024073366A2

WO2024073366A2 - Methods of stabilizing a reducing agent

Info

Publication number: WO2024073366A2
Application number: PCT/US2023/075050
Authority: WO
Inventors: Brittan Pasloske; Richard Crockett
Original assignee: Nuclein, Llc
Priority date: 2022-09-26
Filing date: 2023-09-25
Publication date: 2024-04-04
Also published as: WO2024073366A3

Abstract

The present disclosure relates to methods of stabilizing reducing agents for use in biological methods. These reducing agents include non-thiol based reducing agents that are prepared at acidic pH and/or high concentration. These reducing agents may be used in diagnostics methods that analyze nucleic acids such as PCR.

Description

METHODS OF STABILIZING A REDUCING AGENT The present disclosure claims the benefit of priority to United States Provisional Application No. 63/410,000, filed on September 26, 2022, the entire contents of which are hereby incorporated by reference. BACKGROUND A. FIELD This disclosure relates to methods for sample preparation and treatment useful in biological sciences. More particularly, this disclosure relates to preparing biological samples for in vitro diagnostics. B. RELATED ART Reducing agents have been shown to be useful for molecular biology processes. Pasloske (U.S. Patent No. 6,825,340) claimed the use of reducing agents with heating to inactivate ribonucleases in cell extracts which would protect the RNA in the cell so the RNA was then competent with reverse transcription. In another application, reducing agents are dissolved in water at a high concentration and then added to saliva infected with virus before heating to achieve the desired final concentration (Rabe 2020, Reolo 2021). The saliva sample is then heated to about 90-95 °C for 5 to 10 minutes. This process of heating in the presence of TCEP is thought to inactivate ribonuclease and lyse the virus such that the viral RNA becomes competent for reverse transcription. Reducing agents are frequently used as adjuvants to RNA isolation solutions in conjunction with denaturants to reduce the disulfide bonds in RNases that are rendered accessible by the denaturant. RNase A, RNase 1 and RNase T1 all contain disulfide bonds (Ryle and Anfinesen, 1957; Barnard, 1969) and, therefore, are susceptible to reduction. In biochemistry, reducing agents are also used to stabilize free sulfhydryl groups in proteins and other reagents. Additionally, heat has been used to inactivate RNase A by mediating the breakage of disulfide bonds. Zale and Klibanov (1986) performed inactivation of RNase A at 90 °C and pH 6.0 for 1 hour, which induced the following chemical changes: disulfide interchange, β- elimination of cysteine residues, and deamidation of asparagine. Some reagents can be used as a catalyst increase the rate of reduction. Singh and Kats (1995) demonstrated that selenol increased the reduction rate by as much as 90-fold.

{01069559} 1 4862-4193-3440, v.1 Reducing agents suspended in water oxidize over time (Han 1994) and lose their reducing potential. Typically, to minimize oxidation, reducing agents must be kept frozen to maintain their reducing strength, thawed when needed and then refrozen. This instability is undesirable with respect to developing room temperature stable products designed to process biological samples such that the samples are compatible with biochemical reactions such as reverse transcription. TCEP and THPP are stabler than other reducing agents such as DTT in liquid form (McNulty 2015) and while they are stabler at lower pH’s, they remain susceptible to oxidation in liquid form (Han 1994). Therefore, there is a need to develop ways to improve the stabilization of reducing agents for use in biological applications. SUMMARY Briefly, the present disclosure provides methods, systems, and approaches for the preparation of biological samples comprising a reducing agent and heat. In some aspects, the present disclosure provides methods of preparing a dried form of a reducing agent comprising: (A) obtaining a solution comprising the reducing agent, wherein the concentration of the reducing agent in the solution is greater than 20 mM; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the reducing agent. In still yet another aspect, the present disclosure provides methods of preparing a dried form of an organic reducing agent comprising: (A) obtaining a solution comprising an organic reducing agent; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the reducing agent. In yet another aspect, the present disclosure provides methods of preparing a dried form of a tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP), or a salt thereof comprising: (A) obtaining a solution of TCEP, THPP, or a salt thereof; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the TCEP, THPP, or salt thereof.

{01069559} 2 4862-4193-3440, v.1 In some embodiments, the reducing agent is an organic reducing agent. In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP), tris(3- hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT). In some embodiments, the reducing agent is a salt of a reducing agent. In some embodiments, the salt of the reducing agent is a hydrochloride salt. In some embodiments, the salt of the reducing agent is TCEP hydrochloride. In some embodiments, the methods comprise a solution with a concentration of the reducing agent of greater than 10 mM. In some embodiments, the concentration of the reducing agent is greater than 20 mM. In some embodiments, the concentration of the reducing agent is greater than 40 mM. In some embodiments, the concentration of the reducing agent is greater than 50 mM. In some embodiments, the concentration of the reducing agent is from about 20 mM to about 300 mM. In some embodiments, the concentration of the reducing agent is from about 35 mM to about 200 mM. In some embodiments, the concentration of the reducing agent is from about 50 mM to about 100 mM. In some embodiments, the solution has an acidic pH. In some embodiments, the solution has a pH of less than 6. In some embodiments, the solution has a pH of less than 5. In some embodiments, the solution has a pH from about 2 to about 6. In some embodiments, the solution has a pH from about 3 to about 5. In some embodiments, the solution has a pH of about 4. In some embodiments, the reducing agent further comprises a catalyst. In some embodiments, the catalyst is a selenol such as selenocystamine or selenocystamine dihydrochloride. In some embodiments, the methods comprise applying a fixed volume to the surface. In some embodiments, the fixed volume is applied in one spot on the surface. In other embodiments, the fixed volume is applied to two or more spots on the surface. In some embodiments, the fixed volume of reducing agent is applied in a different spot than the catalyst on the surface. In some embodiments, the fixed volume is from about 0.5 µL to about 50 µL. In some embodiments, the fixed volume is from about 1 µL to about 25 µL. In some embodiments, the fixed volume is from about 2 µL to about 10 µL.

{01069559} 3 4862-4193-3440, v.1 In some embodiments, the surface is a sample preparation vessel. In some embodiments, the sample preparation vessel is a plastic surface. In other embodiments, the sample preparation vessel is a glass surface. In other embodiments, the sample preparation vessel is a metal surface. In some embodiments, the methods comprise drying at a temperature greater than −80 °C. In some embodiments, the methods comprise drying at a temperature greater than 0 °C. In some embodiments, the temperature is greater than 10 °C. In some embodiments, the temperature is from about 0 °C to about 100 °C. In some embodiments, the temperature is from about 10 °C to about 80 °C. In some embodiments, the temperature is from about 20 °C to about 60 °C. In some embodiments, the drying is carried out at a pressure greater than 0.75 atm. In some embodiments, the drying is carried out at a pressure greater than 0.9 atm. In some embodiments, the drying is carried out at a pressure greater than 1 atm. In some embodiments, the solution is an aqueous solution. In some embodiments, the solutions comprise one or more acid or base addition salts. In some embodiments, the solutions comprise an acid addition salt. In other embodiments, the solutions comprise a base addition salt. In some embodiments, the solutions comprise no other compounds other than a solvent, the reducing agent, and one or more acid or base addition salts. In some embodiments, the solutions comprise no other compounds other than a solvent and the reducing agent. In some aspect, the present disclosure provides solid compositions of a reducing agent prepared described herein. In another aspect, the present disclosure provides sample preparation vessels comprising: (A) a sample preparation vessel; and (B) a reducing agent; wherein the reducing agent has been affixed to an internal surface of the sample preparation vessel and wherein the reducing agent is present as a solid. In some embodiments, the reducing agent has been prepared as described herein. In some embodiments, the sample preparation vessel is a conical vial. In still another aspect, the present disclosure provides methods of preparing a raw sample comprising: (A) exposing a raw sample from a patient to a surface comprising a solid reducing agent prepared according to the methods described herein to form a reaction composition; and

{01069559} 4 4862-4193-3440, v.1 (B) heating the reaction composition to obtain a sample for analysis. In some embodiments, the raw sample is a raw sample from a patient. In some embodiments, the patient is a mammal such as a human. In some embodiments, the methods comprise resuspending the reducing agent in a solution. In some embodiments, the raw sample is a biological sample such as saliva, blood, or urine. In some embodiments, the biological sample is saliva. In some embodiments, the methods further comprise analyzing the sample by PCR. In some embodiments, the PCR is RT-PCR. In yet another aspect, the present disclosure provides methods of detecting an abnormal state in a patient comprising: (A) exposing a raw sample from the patient to a reducing agent, wherein the reducing agent has been prepared as described herein to obtain a lysate solution; (B) conducting PCR on the lysate solution to determine the presence of an abnormal state in the patient. In some embodiments, the raw sample is saliva. In some embodiments, the abnormal state is the presence of a disease or disorder. In some embodiments, the abnormal state is an infection of a microorganism. In some embodiments, the abnormal state is an infection of a respiratory pathogen. In some embodiments, the abnormal state is an infection of a virus. In some embodiments, the virus is a coronavirus such as the virus that causes SARS-Cov2. In some embodiments, the methods determine that a patient has an infection of SARS- Cov2. In some embodiments, the PCR is RT-PCR. In some embodiments, the PCR detects the presence of a foreign polynucleotide. In still yet another aspect, the present disclosure provides methods of preparing a sample comprising: (A) obtaining a sample preparation vessel comprising dried reducing agent prepared as described herein; (B) adding a raw sample to the sample preparation vessel; and (C) heating the sample preparation vessel to obtain the sample. Any embodiment of any of the present methods, composition, kit, and systems may consist of or consist essentially of - rather than comprise/include/contain/have - the described steps and/or features. Thus, in any of the claims, the term “consisting of” or “consisting essentially of” may be substituted for any of the open-ended linking verbs recited above, in

{01069559} 5 4862-4193-3440, v.1 order to change the scope of a given claim from what it would otherwise be using the open- ended linking verb. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” or “approximately” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. Following long-standing patent law, the words “a” and “an,” when used in conjunction with the word “comprising” in the claims or specification, denotes one or more, unless specifically noted. As used herein, the terms “comprises,” “comprising,” "includes," "including," "has," "having," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, product, article, or apparatus that comprises a list of elements is not necessarily limited only those elements but may include other elements not expressly listed or inherent to such process, process, article, or apparatus. Furthermore, the term "or" as used herein is generally intended to mean "and/or" unless otherwise indicated. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). As used herein, including the claims that follow, a term preceded by "a" or "an" (and "the" when antecedent basis is "a" or "an") includes both singular and plural of such term, unless clearly indicated within the claim otherwise (i.e., that the reference "a" or "an" clearly indicates only the singular or only the plural). Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. As used herein, "patient" or "subject" includes mammalian organisms, such as human and non-human mammals, for example, but not limited to, rodents, mice, rats, non-human primates, companion animals such as dogs and cats as well as livestock, e.g., sheep, cow, horse, etc. Therefore, for example, although the described embodiments illustrate use of the present methods on humans, those of skill in the art would readily recognize that these methods and compositions could also be applied to veterinary medicine as well as on other animals.

{01069559} 6 4862-4193-3440, v.1 DETAILED DESCRIPTION OF THE INVENTION In certain aspects, the present disclosure provides methods of stably manufacturing, storing or preserving reducing agents. In some embodiments, the reducing agent may be a phosphine, monothiol, dithiol, or inorganic reducing agent. A non-limiting list of reducing agents include those described in, for example, Hopkins Nature, 1930, which is hereby incorporated by reference. These reducing agents may be applied to one or more sample containers to allow for sample preparation. The reducing agent may be affixed to a surface and then stored. These containers may be for part of a kit that can further include one or more other agents and/or instructions for their use. I. Reducing Agents Reducing agents are elements or compounds that donate an electron to an oxidizer compound, hence a compound may be “reduced” (lose an electron) to create an “oxidized” state and the reaction can typically be reversed by “oxidizing” (donating an electron) a compound back into its “reduced” state. At the protein level, reducing agents are often critical in cleaving the disulfide bonds between cysteine amino acids. Commonly used thiol-based reducing reagents are β-mercaptoethanol, 2,3-bis (mercaptoethyl) pyrazine (BMMP), dithiothreitol (DTT), dithioerythritol (DTE), glutathione and cysteine. β-mercaptoethanol is often included in RNA isolation solutions combined with guanidinium thiocyanate to reduce ribonuclease activity and solubilize proteins (Chomcyznski and Sacchi, 1987). There is a second class of “non-thiol” based reducing agents that include TRIS (2-carboxyethyl) phosphine, hydrochloride (TCEP) and TRIS (3-hydroxypropyl) phosphine (THPP). These non-thiol based reducing agents are less susceptible to oxidation and have greater stability than the thiol-based reducing agents. Many reducing reagents are well known to those skilled in the art, and, by employing assays described herein, one of ordinary skill in the art will be able to determine which of any of these reducing agents will be of use in the present invention. Presently preferred reducing agents are those comprising TCEP, THPP, DTT, β-mercaptoethanol, and cysteine. In some embodiments, the reducing agent may be a phosphine, monothiol, dithiol, or inorganic reducing agent. A non-limiting list of reducing agents include those described in, for example, Hopkins Nature, 1930, which is hereby incorporated by reference. In one particular

{01069559} 7 4862-4193-3440, v.1 embodiment, the reducing agent may be TCEP (tris-(2-carboxyethyl) phosphine), THPP (tris(3-hydroxypropyl)phosphine), BMMP (2,3-bis(mercaptomethyl)pyrazine), GSH (glutathione), dithiothreitol (DTT), or dithioerythritol (DTE). In other embodiments, the reducing agent may be TBP (tributyl phosphine), THMP (tris-(hydroxymethyl) phosphine), DHAA (dihydroasparagusic acid), BMS (bis(2-mercaptoethyl) sulfone), Meso-DTA (meso- 2,5-dimercapto-N,N,N’,N’-tetramethyladipamide), DMH (N,N’-dimethyl-N,N’- bis(mercaptoacetyl)hydrazine), DTBA ((2S)-2-amino-1,4-dimercaptobutane), DABDT (2,3- bis(mercaptomethyl)pyrazine), Sodium borohydride, Zn/H+ or Sn/H+. i. Ellman’s Assay to Measure Reducing Potential In order to determine the efficacy of a reducing agent, a method of determining the reducing agent’s activity is needed. The most common way to determine this activity or reducing potential is Ellman’s Assay (Ellman 1958). Reducing activity can be accurately assayed using 5, 5'-dithiobis (2-nitrobenzoic acid) or DTNB, also known as Ellman’s reagent (Ellman 1959). The reduction of DTNB mediated by a reducing agent generates a yellow color whose absorbance can be measured at 412 nm using a spectrophotometer. The Ellman’s assay is used to measure the reducing potential of reducing agents such as TCEP and THPP. The standard assay used to measure reducing potential was as follows. DNTB is dissolved to 0.08 mg/ml in 100 mM TRIS pH 8.0: 1 mM EDTA pH 8.0 (TE) to produce Ellman’s solution. TCEP standards were stored at -20°C to maximize their stability. They were thawed and diluted to 0.5 mM just prior to use. To assay the reducing potential of the test samples, the samples were diluted in water to generate an expected TCEP concentration of 0.5 mM. To assay each sample, 10 uL of the sample (at the expected 0.5 mM concentration) was pipetted into the well of a clear, flat-bottomed 96-well plate. After all samples were pipetted, 200 µL of Ellman’s solution was pipetted to each sample and pipetted up and down several times to mix. The 96-well plate was then placed in a spectrophotometer and the samples read at a wavelength of 412 nm. The absorbances of the samples were compared to the absorbances of the TCEP standard. The results are usually reported as a percent of the TCEP standard stored at -20°C. II. Method of Stabilizing the Reducing Agent In some embodiments, the reducing agent, which may optionally include a catalyst, is in a shelf-stable format. As used herein, “shelf-stable” means that a reagent, formulation, or

{01069559} 8 4862-4193-3440, v.1 manufacturing process maintains at least 50% of its original activity at a temperature above about 10°C for a period of at least 72 hours. As used herein, “about” means that a description may vary by ± 10%. The present disclosure presents methods of preparing these shelf-stable reducing agents including taking a solution with the reducing agent at an acidic pH and then drying the solution In some embodiments, the reducing agent and/or catalyst are shelf-stable. In particular embodiments, the shelf-stable reducing agent and/or catalyst are additionally stable at a temperature above about 10ºC, 15°C, 20°C, 25°C, 40°C, 45°C, 50°C, 60°C, 70°C, 80°C, 90°C, 95°C or higher. In certain embodiments, the shelf-stable reducing agent and/or catalyst are additionally stable at a humidity above about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In certain embodiments, the reducing agent may be in a shelf-stable solid form, optionally including a catalyst. In particular embodiments, the concentration of the reducing agent for application to a surface may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM or higher. The concentration of the reducing agent may be from about 1 mM to about 500 mM, from about 5 mM to about 250 mM, or from about 25 mM to about 100 mM. The concentration of the reducing agent may be from about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, to about 1 M, or any range derivable therein. In particular embodiments the pH of the reducing agent and or catalyst is less than about 8, 7, 6, 5, 4, 3, 2, or 1. The pH of the solution containing the reducing agent may be less than 8, 7, 6, 5, 4, 3, 2, or 1. The pH of the solution containing the reducing agent may be from about 1 to about 8, from about 2 to about 6, from about 3 to about 5. The pH may be from about 1, 2, 3, 4, 5, 6, 7, to about 8, or any range derivable therein. In some embodiments, the reducing agent may be applied in liquid, droplet, or vapor form to a surface. In particular embodiments, the applied reducing agent may dried on the surface. In some embodiments, the atmospheric pressure during the drying process may be greater than about 0 atm, 0.1 atm, 0.25 atm, 0.5 atm, 0.75 atm, 1 atm, or higher. In particular, the pressure may be from about 0 atm to about 5 atm, from about 0.1 atm to about 2.5 atm, or

{01069559} 9 4862-4193-3440, v.1 from about 0.25 atm to 1 atm. The pressure may be from about 0.1 atm, 0.2 atm, 0.25 atm, 0.3 atm, 0.4 atm, 0.5 atm, 0.6 atm, 0.7 atm, 0.8 atm, 0.9 atm, 1.0 atm, 1.25 atm, 1.5 atm, 1.75 atm, 2.0 atm, 2.5 atm, 3.0 atm, 3.5 atm, 4.0 atm, 4.5 atm, to about 5 atm, or any range derivable therein. In particular embodiments, the temperature during the drying process may be above about −80°C, −75°C, −70°C, −65°C, −60°C, −55°C, −50°C, −45°C, −40°C, −35°C, −30°C, −25°C, −20°C, −15°C, −10°C, −5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 99°C, or higher. Similarly, the temperature of the drying process can be from about 0°C to about 100°C, from about 5°C to about 75°C, or from about 10°C to about 50°C. The temperature may be from about −80°C, −75°C, −70°C, −65°C, −60°C, −55°C, −50°C, −45°C, −40°C, −35°C, −30°C, −25°C, −20°C, −15°C, −10°C, −5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, to about 100°C, or any range derivable therein. In some embodiments, the reducing agent is dried at a temperature higher than ambient temperature and/or a pressure lower than ambient pressure to accelerate the drying process. III. Kits for Biological Analysis The present disclosure contemplates kits that may be used to prepare biological samples. The stabilized reducing agent may be coupled with one or more additives or pre- packaged in a collection tube to allow the ready processing of a raw sample. The kit may further comprise one or more further active ingredients and/or instructions for the use of the reducing agents. The reducing agent may be affixed to the surface of a sample container that is contained within the kit to form a usable sample. In one embodiment, the reducing agent is resuspended in water or a buffer, then mixed with the raw sample and heated. In another embodiment, the dried reducing agent is resuspended by the raw sample and heated. In another embodiment, the heated sample is subjected to PCR or RT-PCR. In certain embodiments, the reducing agent is TCEP, THPP or DTT. In certain embodiments, the reducing agents prepared as described herein may be used to prepare a raw sample for analysis. The raw sample may be a liquid sample. In other embodiments, the liquid raw sample is saliva, urine, mucous, sputum, phlegm, blood, tears,

{01069559} 10 4862-4193-3440, v.1 cerebral spinal fluid (CSF), breast milk, pus, sebum, semen, smegma, pustule discharge, vaginal secretions, feces, synovial fluid, bile, gastric fluid, chyme, amniotic fluid, lochia, vitreous fluid, ear wax and perspiration. In other embodiments, the liquid raw sample is generated by swabbing a surface, incubating the swab in a liquid for a period of time and then adding the liquid to the dried reducing agent. In other embodiments, the reducing agent is resuspended by water or a buffer and the swab immersed directly in the resuspended reducing agent. In some embodiments the shelf-stable reducing agent and/or any other component comprised are dried on a surface with a volume less than about 500 µL, 250 µL, 100 µL, 75 µL, 50 µL, 25 µL, 15 µL, 10 µL, 9 µL, 8 µL, 7 µL, 6 µL, 5 µL, 4 µL, 3 µL, 2 µL, or 1 µL. The amount of the shelf stable reducing agent or other component may be from about 1 µL to about 500 µL, from about 2.5 µL to about 100 µL, from about 5 µL to about 50 µL. The amount may be from about 1 µL, 2.5 µL, 5 µL, 7.5 µL, 10 µL, 15 µL, 20 µL, 25 µL, 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, 80 µL, 90 µL, 100 µL, 150 µL, 200 µL, 300 µL, 400 µL, to about 500 µL, or any range derivable therein. In some embodiments, the reducing agent may be applied in liquid, droplet, or vapor form to a surface. In particular embodiments, the applied reducing agent may dried on the surface. In another embodiment, the reducing agent is applied to a surface using a spin coat method. In certain embodiments, the surface is a polymer. In other embodiments, the surface is a metal or glass. In still other embodiments, the surface may be an organic material. In specific embodiments, the surface may be a flat or curvilinear surface. In other embodiments the surface may comprise one or more wells or vessels for sample preparation. In particular embodiments, the surface may be porous or comprise a filter material. In certain embodiments, the surface may include features to facilitate mixing or turbulence of a fluid, capture of the reducing agent and/or catalyst prior to drying, or contain features to alter the probability of mixing, for example keeping the reducing agent and catalyst separate, or conversely, increasing the probability that they will combine prior to or during drying, or subsequent to drying, for example, during resuspension or dissolution. In additional embodiments, the surface polymer exhibits a low surface energy that decreases the ratio of surface area to volume of the dried reducing agent and/or catalyst. In

{01069559} 11 4862-4193-3440, v.1 other embodiments, the polymer exhibits a high surface area that increases the ratio of surface area to volume of the dried reducing agent and/or catalyst. In particular embodiments, the ratio of surface area to volume affects the drying and resuspension properties of the reducing agent and/or catalyst. In specific embodiments, a low surface area to volume ratio minimizes the exposure of the reducing agent to oxidation, which may be useful to a practitioner to extend the shelf-stability of the disclosure. In other embodiments, a high surface area to volume ratio of the reducing agent and/or catalyst may increase the rate of resuspension or dissolution, or may increase the fraction that is resuspended or dissolved, which may be useful to a practitioner to decrease the amount of time or increase the efficiency of the disclosure. In certain embodiments, the polymer is a polypropylene. In specific embodiments, the polypropylene is a co-polymer. In other embodiments the polypropylene is a homopolymer. In some embodiments, the polymer may be polyethylene terephthalate (PET), polystyrene, polyoxymethylene copolymer (POM) and polyvinyl styrene (PVC). In certain embodiments, the reducing agent may be in a shelf-stable solid form, optionally including another component such as a catalyst. In particular embodiments, the concentration of the reducing agent for application to a surface may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM or higher. The concentration of the reducing agent may be from about 1 mM to about 500 mM, from about 5 mM to about 250 mM, or from about 25 mM to about 100 mM. The concentration of the reducing agent may be from about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, to about 1 M, or any range derivable therein. In some embodiments, the concentration of the other components may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM or higher. The concentration of the other components may be from about 1 mM to about 500 mM, from about 5 mM to about 250 mM, or from about 25 mM to about 100 mM. The concentration of the other components may be from about 1 mM, 5 mM, 10 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, to about 1 M, or any range derivable therein.

{01069559} 12 4862-4193-3440, v.1 In particular embodiments the pH of the reducing agent and or catalyst is less than about 8, 7, 6, 5, 4, 3, 2, or 1. The pH of the solution containing the reducing agent may be less than 8, 7, 6, 5, 4, 3, 2, or 1. The pH of the solution containing the reducing agent may be from about 1 to about 8, from about 2 to about 6, from about 3 to about 5. The pH may be from about 1, 2, 3, 4, 5, 6, 7 to about 8, or any range derivable therein. In particular embodiments, a catalyst or other component may be included with the reducing agent. In certain embodiments, the catalyst or other component may be mixed with the reducing agent before a drying process. In other embodiments, the catalyst or other component and reducing agent may be independently applied to the surface without mixing. In some embodiments, the reducing agent and catalyst or other component are dried on different areas of the same surface. In other embodiments the reducing agent and catalyst or other component are dried on different areas of different surfaces. In particular embodiments, the dried reducing agent and catalyst or other component are subsequently resuspended or dissolved in a liquid. In certain particular embodiments, the reducing agent and catalyst or other component are mixed after a drying process while suspended or dissolved in a liquid. In specific embodiments, one or more catalysts may be included with the reducing agent. In particular embodiments, the catalyst may comprise a selenol. In even more particular embodiments, the catalyst may be selenocystamine or selenocystamine dihydrochloride. In some embodiments, the catalyst may be a chaotrope. In particular embodiments, the chaotrope may be a hydrogen bond competitor, polar destabilizer, or detergent. In some embodiments, the chaotrope may be guanidine, urea, thiourea, lithium perchlorate, lithium acetate, or an alcohol, for example, ethanol, n-butanol, isopropyl, or other alcohols which will be apparent to the person of ordinary skill. In certain embodiments, the detergent may be SDS (sodium dodecyl sulfate), Triton X100, Brij, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), glycine, proline, octylglucoside, digitonin, or other detergents that will be apparent to the practitioner.

{01069559} 13 4862-4193-3440, v.1 IV. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 – Stabilization of Reducing Agents A. TCEP Stability at Different pH’s and Temperatures TCEP solutions were prepared to 50 mM at pH 4, 7 and 9. For each of these samples, 4 µL was spotted to the bottom of glass vials, dried to completion at 40°C and then capped with rubber stoppers. The vials were incubated at room temperature (~21°C), 40 °C and 55°C. After 10 days, each spot was assayed using the Ellman’s assay using the frozen Time “0” sample as the control. Results are reported as a percent of the Time 0 control.

From these results, it was observed that there was decreasing stability with increasing temperature and pH. However, at pH 4, TCEP was stable at all three temperatures. B. Stability Study of Dried Down TCEP The stability of dried down TCEP on polypropylene cassettes was evaluated to demonstrate the feasibility of the drying process. 8 µL 50 mM TCEP (pH 4.0) was pipetted into the wells of the polypropylene cassettes. The TCEP spots were dried at 40°C for about 4 hours. The cassettes were then moved to nitrogen cabinets to purge the air from the cassettes. The cassettes were then individually placed into 4 inch × 6 inch mylar bags with one desiccant bag. The bags were squeezed open and close a few times to exchange air with nitrogen. Subsequently, the bags were heat sealed to prevent any air from exchanging into the bag.

{01069559} 14 4862-4193-3440, v.1 The bags were then stored at one of three temperatures: room temperature (~20°C), 40°C and 55°C. The dried TCEP in the cassettes was periodically assayed to measure the reducing potential to assess stability. The higher temperatures were included to accelerate the stability study and to mimic transport at higher temperatures. After 43 days, the dried-down TCEP was very stable. At 55°C, the reducing potential of the TCEP was 98% of the control which is the equivalent of about 14 months at 21°C. The samples at room temperature and 40°C remained at about 100% of the control. C. THPP Stability In a separate experiment following the same procedures, a stability study was performed for dried-down THPP (pH 4.0) on polypropylene cassettes in nitrogen. After 20 days, it was observed that the THPP maintained 100% of its reducing potential at all three incubation temperatures. At 55°C for 18 days is the equivalent of more than 6 months at 21°C. D. TCEP and THPP Stability in High Humidity 8 µL of 50 mM TCEP (pH 4.0) and 50 mM THPP were pipetted onto polypropylene cassettes. They were incubated at 40ºC until the spots were dry which was about 3.5 h. The cassettes were put into mylar bags with a damp cloth and a humidity logger and then sealed. The cassettes were stored at room temperature for 5 days. The humidity loggers indicated that the humidity for the incubation period was at least 90%. Afterwards, the samples were assayed using the Ellman’s assay. The samples maintained 100% reducing potential, even at these high humidity conditions in the presence of ambient air. E. Preparing a Saliva Sample for RT-PCR 3 µL of 50 mM TCEP (pH 4.0) was pipetted into the bottom of 1.5 mL microfuge tubes and dried overnight at room temperature (~20°C). A few milliliters (mL) of saliva from 4 different donors was collected by drooling over different days. The saliva was spiked with gamma-irradiated SARS-CoV-2 (from BEI) to a concentration of 1 × 10³ copies per mL of saliva. Quantities of 150 µL of the spiked saliva were pipetted into the TCEP tubes. The tubes were incubated 90°C for 7 minutes and then removed from the heat to room temperature.

{01069559} 15 4862-4193-3440, v.1 The heat lysed saliva was added to RT-PCR reactions to one-tenth the volume of the total reactions. These PCR reactions targeted SARS-CoV-2 using the Nuclein® amplification chamber. In a total of 26 different reactions, over 4 different experiments, a strong positive SARS-CoV-2 signal was produced. F. Resuspension of TCEP Prior to Mixing with Saliva 8 µL of 50 mM TCEP (pH 4.0) was pipetted in the bottom of 1.2 mL flat-bottom plastic wells and dried overnight at room temperature (~20°C). 400 µL of water was added to the well with TCEP and allowed to sit for 2 minutes.600 µL of saliva spiked with 3 × 10³ copies per mL of gamma irradiated SARS-CoV-2 was added to the water and pipetted up and down several times to mix.0.5 ml of the mixture was pipetted to a 1.5 mL microfuge tube and then incubated at 90°C for 7 minutes. 150 µL of the heat lysed saliva was added to 800 µL of water and pipetted up and down several times to mix. This mixture was then dispensed to Nuclein® amplification chambers containing lyophilized beads for RT-PCR to detect SARS-CoV-2. The material in the chambers was progressed through RT-PCR. In all cases, the SARS-CoV-2 was detected. G. Spin Coating Spin coating is a common technique for applying thin films to substrates. When a solution of a material and a solvent is spun at high speeds, the centripetal force and the surface tension of the liquid together create an even covering. After any remaining solvent has evaporated, spin coating results in a thin film ranging from a few nanometres to a few microns in thickness. Spin coating is used in a wide variety of industries and technology sectors. Its primary advantage of spin coating over other methods is its ability to quickly and easily produce very uniform films. * * * * * All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the

{01069559} 16 4862-4193-3440, v.1 sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

{01069559} 17 4862-4193-3440, v.1

Claims

WHAT IS CLAIMED: 1. A method of preparing a dried form of a reducing agent comprising: (A) obtaining a solution comprising the reducing agent, wherein the concentration of the reducing agent in the solution is greater than 20 mM; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the reducing agent.

2. A method of preparing a dried form of an organic reducing agent comprising: (A) obtaining a solution comprising an organic reducing agent; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the reducing agent.

3. A method of preparing a dried form of a tris(2-carboxyethyl)phosphine (TCEP), tris(3- hydroxypropyl)phosphine (THPP), or a salt thereof comprising: (A) obtaining a solution of TCEP, THPP, or a salt thereof; (B) applying the solution to a surface; and (C) drying the surface at a pressure of at least 0.5 atm to obtain the dried form of the TCEP, THPP, or salt thereof.

4. The method of claim 1, wherein the reducing agent is an organic reducing agent.

5. The method according to any one of claims 1, 2, or 4, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT).

6. The method according to any one of claims 1-5, wherein the reducing agent is a salt of a reducing agent.

7. The method of claim 6, wherein the salt of the reducing agent is a hydrochloride salt.

8. The method of either claim 6 or claim 7, wherein the salt of the reducing agent is TCEP hydrochloride.

9. The method according to any one of claims 2-8, wherein the method comprises a solution with a concentration of the reducing agent of greater than 10 mM.

10. The method according to any one of claims 2-9, wherein the concentration of the reducing agent is greater than 20 mM.

11. The method according to any one of claims 1-10, wherein the concentration of the reducing agent is greater than 40 mM.

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12. The method according to any one of claims 1-11, wherein the concentration of the reducing agent is greater than 50 mM.

13. The method according to any one of claims 1-11, wherein the concentration of the reducing agent is from about 20 mM to about 300 mM.

14. The method according to any one of claims 1-13, wherein the concentration of the reducing agent is from about 35 mM to about 200 mM.

15. The method according to any one of claims 1-14, wherein the concentration of the reducing agent is from about 50 mM to about 100 mM.

16. The method according to any one of claims 1-15, wherein the solution has an acidic pH.

17. The method according to any one of claims 1-16, wherein the solution has a pH of less than 6.

18. The method according to any one of claims 1-17, wherein the solution has a pH of less than 5.

19. The method according to any one of claims 1-17, wherein the solution has a pH from about 2 to about 6.

20. The method according to any one of claims 1-19, wherein the solution has a pH from about 3 to about 5.

21. The method according to any one of claims 1-20, wherein the solution has a pH of about 4.

22. The method according to any one of claims 1-21, wherein the reducing agent includes a catalyst.

23. The method of claim 22, wherein the catalyst is a selenol.

24. The method of claim 23, wherein the selenol is selenocystamine or selenocystamine dihydrochloride.

25. The method according to any one of claims 1-24, wherein the method comprises applying a fixed volume to the surface.

26. The method of claim 25, wherein the fixed volume is applied in one spot on the surface.

27. The method of claim 25, wherein the fixed volume is applied to two or more spots on the surface.

28. The method according to any one of claims 1-27, wherein the fixed volume of reducing agent is applied in a different spot than the catalyst on the surface.

29. The method according to any one of claims 25-28, wherein the fixed volume is from about 0.5 µL to about 50 µL.

30. The method according to any one of claims 25-29, wherein the fixed volume is from about 1 µL to about 25 µL.

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31. The method according to any one of claims 25-30, wherein the fixed volume is from about 2 µL to about 10 µL.

32. The method according to any one of claims 1-31, wherein the surface is a sample preparation vessel.

33. The method of claim 32, wherein the sample preparation vessel is a plastic surface.

34. The method of claim 32, wherein the sample preparation vessel is a glass surface.

35. The method of claim 32, wherein the sample preparation vessel is a metal surface.

36. The method according to any one of claims 1-35, wherein the method comprises drying at a temperature greater than −80 °C.

37. The method according to any one of claims 1-34, wherein the method comprises drying at a temperature greater than 0 °C.

38. The method according to any one of claims 1-37, wherein the temperature is greater than 10 °C.

39. The method according to any one of claims 1-38, wherein the temperature is from about 0 °C to about 100 °C.

40. The method according to any one of claims 1-39, wherein the temperature is from about 10 °C to about 80 °C.

41. The method according to any one of claims 1-40, wherein the temperature is from about 20 °C to about 60 °C.

42. The method according to any one of claims 1-41, wherein the drying is carried out at a pressure greater than 0.75 atm.

43. The method according to any one of claims 1-42, wherein the drying is carried out at a pressure greater than 0.9 atm.

44. The method according to any one of claims 1-43, wherein the drying is carried out at a pressure greater than 1 atm.

45. The method according to any one of claims 1-44, wherein the solution is an aqueous solution.

46. The method according to any one of claims 1-45, wherein the solution comprises one or more acid or base addition salts.

47. The method according to any one of claims 1-46, wherein the solution comprises an acid addition salt.

48. The method according to any one of claims 1-46, wherein the solution comprises a base addition salt.

49. The method according to any one of claims 1-48, wherein the solution comprises no other compounds other than a solvent, the reducing agent, and one or more acid or base addition salts.

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50. The method according to any one of claims 1-49, wherein the solution comprises no other compounds other than a solvent and the reducing agent.

51. A solid composition of a reducing agent prepared according to the methods in claims 1-50.

52. A sample preparation vessel comprising: (A) a sample preparation vessel; and (B) a reducing agent; wherein the reducing agent has been affixed to an internal surface of the sample preparation vessel and wherein the reducing agent is present as a solid.

53. The sample preparation vessel of claim 52, wherein the reducing agent has been prepared according to any one of claims 1-50.

54. The sample preparation vessel of either claim 52 or claim 53, wherein the sample preparation vessel is a conical vial.

55. A method of preparing a sample comprising: (A) exposing a raw sample from a patient to a surface comprising a solid reducing agent prepared according to the methods of claims 1-50 to form a reaction composition; and (B) heating the reaction composition to obtain a sample for analysis.

56. The method of claim 55, wherein the raw sample is a raw sample from a patient.

57. The method of claim 56, wherein the patient is a mammal.

58. The method of claim 57, wherein the mammal is a human.

59. The method according to any one of claims 55-58, wherein the method comprises resuspending the reducing agent in a solution.

60. The method according to any one of claims 55-59, wherein the raw sample is a biological sample.

61. The method of claim 60, wherein the biological sample is saliva, blood, or urine.

62. The method of either claim 60 or claim 61, wherein the biological sample is saliva.

63. The method according to any one of claims 55-62, wherein the method further comprises analyzing the sample by PCR.

64. The method of claim 63, wherein the PCR is RT-PCR.

65. A method of detecting an abnormal state in a patient comprising: (A) exposing a raw sample from the patient to a reducing agent, wherein the reducing agent has been prepared as described in claims 1-51 to obtain a lysate solution;

{01069559} 21 ^{4862-4193-3440, v. 1} (B) conducting PCR on the lysate solution to determine the presence of an abnormal state in the patient.

66. The method of claim 65, wherein the raw sample is saliva.

67. The method of either claim 65 or claim 66, wherein the abnormal state is the presence of a disease or disorder.

68. The method according to any one of claims 65-67, wherein the abnormal state is an infection of a microorganism.

69. The method according to any one of claims 65-68, wherein the abnormal state is an infection of a respiratory pathogen.

70. The method according to any one of claims 65-69, wherein the abnormal state is an infection of a virus.

71. The method of claim 70, wherein the virus is a coronavirus.

72. The method of claim 71, wherein the coronavirus is the virus that causes SARS-Cov2.

73. The method according to any one of claims 65-72, wherein the method determines that a patient has an infection of SARS-Cov2.

74. The method according to any one of claims 65-73, wherein the PCR is RT-PCR.

75. The method according to any one of claims 65-74, wherein the PCR detects the presence of a foreign polynucleotide.

76. A method of preparing a sample comprising: (A) obtaining a sample preparation vessel comprising dried reducing agent prepared as described in claims 1-51; (B) adding a raw sample to the sample preparation vessel; and (C) heating the sample preparation vessel to obtain the sample.

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