Biomolecules isolation method and devices using a cloudy precipitated solution

Abstract

The invention relates to a unified system for the extraction and purification of biomolecules using a novel Cloudy-Precipitated (CP) Buffer and an integrated extraction device. The CP Solution remains cloudy even when heated and enables single- or multi-step workflows for isolating nucleic acids without requiring multiple buffers or organic solvents. It contains optimized concentrations of detergents, chaotropic agents, salts, and adsorbents to efficiently lyse cells and minimize processing steps. The associated device incorporates a heating and filtration system that allows separation of target molecules from inhibitors or impurities based on molecular weight. It can operate as a single- or double-step configuration with various filter membrane options. This system reduces time, labor, and environmental impact while enhancing sample quality for molecular diagnostics, research, and therapeutic applications.

Description

DescriptionBiomolecules Isolation Method and Devices Using a Cloudy Precipitated Solution

[0001] Field of the Invention this invention relates to methods and devices for extracting biomolecular analytes from a biological sample. The analytes include nucleic acids (DNA, RNA), proteins, peptides, lipids, polysaccharides, and other biological macromolecules. The invention comprises two aspects: (1) a unique Cloudy Precipitated (CP) buffer composition used for the simultaneous extraction and purification of multiple biomolecules, and (2) a modular device that facilitates biomolecule separation using integrated heating, filtration, and sample preparation zones. These systems improve downstream applications such as PCR, sequencing, or metabohte analysis by reducing inhibitors and improving yield and purity.

[0002] Technical Field the invention provides an integrated system involving a buffer and device for the efficient extraction of biomolecules from any biological sample, including but not bmited to human, animal, plant, microbial, and environmental sources. The CP solution and the device are compatible with nucleic acid and protein purification workflows for molecular biology, clinical diagnostics, food safety, agricultural biotechnology, and environmental monitoring.

[0003] The CP solution buffer enables multiphasic, non-homogeneous colloidal conditions facilitating co-extraction of chemically diverse biomolecules with high yield and purity. The associated device is designed to perform one -step or two-step lysis, filtration, and separation using features like heating modules, multi-layer filter stacks, and magnetic or membrane zones to suit various sample types and volumes (e.g., 2 mL, 50 mL formats).

[0004] Background of the Invention Biomolecule extraction is a critical first step in biological assays and diagnostics. Traditional methods for isolating DNA, RNA, proteins, or other macromolecules require separate buffers, multiple steps, long processing times, and the use of toxic reagents or expensive consumables. Thesemethods — such as spin columns, silica membranes, or organic solvent -based techniques — are labor-intensive and often result in inconsistent yields and contamination that interfere with downstream apphcations.

[0005] Nucleic acids (DNA, RNA) serve as key analytes in molecular diagnostics, biotechnology, and research. These molecules occur in various compartments: chromosomal, organellar (mitochondria, chloroplast), plasmids, or cell-free forms in biological fluids. Current extraction methods face hmitations when deahng with complex samples, such as gram -positive bacteria, fungi, or biological fluids containing inhibitors. Many extraction protocols involve costly kits and hazardous waste generation, limiting their use in point-of-care or high-throughput settings.

[0006] This invention addresses these challenges through a novel buffer and an optimized device system: The CP buffer maintains a cloudy, multiphasic state even upon heating or sample addition, improving lysis, stabihzation, and separation of nucleic acids, proteins, and metabolites from the same sample. The device enables modular extraction using either one-step or two-step workflows, reducing time, labor, and contamination risks. It supports sample pre-heating, filtration, and inhibitor removal using integrated chambers with layered separation modules.

[0007] Need for the Invention I Objectives there is a need for an integrated, low- cost, user-friendly biomolecule extraction method that: Reduces time and steps for sample processing. Allows simultaneous extraction of multiple biomolecule classes (DNA, RNA, proteins, etc.). Avoids use of harmful solvents or expensive accessories. Offers consistency and high purity across sample types. Enables direct use in downstream processes like PCR or sequencing.

[0008] The invention offers the following improvements. A CP buffer formulation that enables concurrent extraction of biomolecules under cloudy -phase conditions, acting as both lysis and preservation medium. A modular device with filter stacks, membrane support, and optional heating / magnetic zones, suited for various sample types and compatible with automated or field workflows. A unified method thatensures high-yield extraction even from difficult matrices like stool, blood, saliva, plant tissues, or gram-positive cultures.

[0009] Cloudy Precipitated (CP) Buffer Composition for Simultaneous Extraction and Purification of Biomolecules. An aspect of the invention pertains to the collection and storage of biological samples, and separation of nucleic acids from these biological samples using one or more of a chemical solution(s), the composition of the chemical solutions, the process of using the solutions in a unique order and all the components as a kit, as given here.

[0010] The invention described here is not limited only to the particular sample type or a unique challenge but can be used with flexibility using different combinations of the embodiments. A few examples are shown for making various changes with equivalents or with substitutions without deviating from the scope of the invention.

[0011] The present application does not claim priority from any other patent application(s).

[0012] In some embodiments, the invention relates to a cloudy precipitated (CP) buffer formulation that remains cloudy even when heated and enables one-step minimal sample purification suitable for direct nucleic acid amplification (Figure 1). This CP solution does not become clear upon heating unless organic solvents are added. It can be used effectively in its cloudy state. If needed, further purification can be performed from the crude extract. The invention provides high yield and quality of nucleic acids either in a single step or through a specific sequence of multiple steps using different buffer compositions under optimized conditions. The disclosed buffer compositions are used in a process and kit for nucleic acid extraction. Whether used directly or after purification, the method yields comparable or better quality due to minimal loss of nucleic acids, resulting in high molecular weight fragments.

[0013] Throughout the specification and claims, the terms used have the meanings clearly stated unless the context requires otherwise. The words "a," "an," and "the" also include the plural. The word "in" includes both "in" and "on." In the drawings,the same numbers refer to the same parts. Unless clearly inconsistent, singular terms also include the plural. The terms "CP Solution," "OM Buffer," or "Solutions" refer to lysis buffers with one or more embodiments and compositions.

[0014] An aspect of the invention pertains to a process for the extraction and separation of nucleic acids from a biological sample comprising: The Cloudy- Precipitated Solution serves as a single extraction buffer (lysis as well), where the sample is mixed with the CP solution and centrifuged to separate the nucleic acids from the impurities. The separated nucleic acids can be used for any further application.

[0015] However, the CP solution and the method (One-to Multiple (OM)) are not limited to extraction alone, but can also be used as a collection medium, storage medium, lysis buffer, and extraction buffer based on the application of all molecules.

[0016] Another aspect of the invention relates to the buffer compositions used for extracting and separating nucleic acids. The CP and One-to-Multiple (OM) process includes a unique mix of salts, chaotropic agents, detergents, surfactants, precipitating agents, solvents, and other components. The CP Solution stays cloudy and shows a precipitated appearance both with and without samples, and even when heated, unless organic solvents are added to clear it. Unlike other lysis buffers, this CP Solution can lyse cells and extract nucleic acids and other biomolecules while still in the cloudy, precipitated form. In contrast to prior art, such as US 6762027, where an additional surfactant or extraction agent is required, this method avoids that second step. Also, as noted by Robert E. Farrell, SDS is not commonly used with guanidinium buffers due to its low solubility in high salt, chaotropic conditions.

[0017] There are several embodiments of the CP Solution usedin the One-to-Multiple Process. These include compositions with one or more anionic, cationic, and non-ionic detergents. The anionic surfactants include Cholic Acid, Sodium Dodecyl Sulphate (SDS), their salts, and combinations. The non-ionic surfactants include Polysorbates, Triton, Tweens, Polyoxyethylene Ethers, Phenyl Ethylene Glycols, Ethylene Oxides,and derivatives of Castor Oil such as Castor Oil Ethoxylate, Poly-Hydrogenated Castor Oil, Sulfonated Castor Oil, Ethoxylated Alkyl Phenol (NP-40), Lauryl Alcohol Ethoxylate, Polyoxyethylene (20) Cetyl Ether, and bigCHAP (N,N-Bis[3-(D- Gluconamido)Propyl]-Cholamide). Other components include Sorbitan Monooleate, Sodium Deoxycholate, and various salts such as Sodium Acetate, Potassium Acetate, Ammonium Acetate, Lithium Acetate, Sodium Chloride, Potassium Chloride, Potassium Phosphate, Sodium Citrate, Sodium Salicylates, Diammonium Hydrogen Phosphate, Sodium Phosphate, and Sodium Sulphate.Chaotropic agents include Potassium Iodide, Sodium Thiocyanate, Guanidine Thiocyanate, Urea, Guanidine Carbonate, Guanidine Phosphate, Guanidine Hydrochloride, Sodium Iodide, and Thiourea. Other ingredients may include oils, organic solvents, adsorbents and absorbents such as powdered charcoal, agar, agarose, pectin, gelatin, collagen, PVP, Sodium Azide, and Polyvinyl compounds (like Polyvinyl Chloride, Polyvinyl Acetate, Polyvinylpyrrolidone, Polyvinylpolypyrrolidone), as well as Acrylates and Diethanolamine derivatives (e.g., DEA-Cetyl Phosphate, DEA Oleth-3 Phosphate, Cocamide DEA, Lauramide DEA). Buffers such as TRIS, MOPS, MES, and CHAPS are included. Additional materials can include Toluene derivatives (e.g., Benzyl- and DibenzyLToluene, Butylated Hydroxy Toluene), polystyrene, polyacrylamide, nylon, silica, woodchips, activated charcoal, aluminum oxide, diatomaceous earth (e.g., Cehte), cellulose materials, controlled pore glass, siliconized glass beads, or their combinations.

[0018] Precipitation Solution composition comprising of salts, Guanidinium Isothiocyanate or Guanidinium Hydrochloride, 1-5M of Sodium Acetate or Potassium Acetate, Ammonium Acetate, Lithium Acetate, Sodium Acetate, Sodium Chloride, Potassium Chloride, Tween, Triton, Alcohols.

[0019] Binding Buffer composition comprising anionic, cationic, and non-ionic detergents, salts, chaotropic agents, salts, Guanidinium Isothiocyanate or Guanidinium Hydrochloride, 1-5M of Sodium Acetate or Potassium Acetate,Ammonium Acetate, Lithium Acetate, Sodium Acetate, Sodium Chloride, Potassium Chloride, Tween, Triton, Alcohols.

[0020] Wash Buffer 1 comprising anionic, cationic, and non-ionic detergents, Guanidinium Isothiocyanate or Guanidinium Hydrochloride, 1-5M of Sodium Acetate or Potassium Acetate, Ammonium Acetate, Lithium Acetate, Sodium Acetate, Sodium Chloride, Potassium Chloride, Tween, Triton, Salts, Chaotropic Agents, Salts, Alcohols.

[0021] Wash Buffer 2 comprising a C1-C10 Alcohol. 70% Ethanol, 1 to 250 mM Citrate Buffer, and 10 to 300 mM Phosphate Buffer.

[0022] Elution Buffer comprising Tris, EDTA, EGTA, 1 to 25 mM Tris, 1 to 20 mM EDTA, 1 to 50 mM EGTA.

[0023] Prewash Buffer comprising a C1-C10 Alcohol or its combination is used. 30 % Methanol, 10% Ethanol, 60 % Butanol, and 10 to 300 mM Phosphate Buffer.

[0024] Composition of a model CP Solution but not limited to O.lx to lOx concentrated buffer of the below can be used as follows: 1 to 25 mM Tris, 1 to 30 mM EDTA, 1 to 50 mM EGTA, 0.01% to 5 % CTAB, 0.1 to 6% SDS,10 to 1000 mM Sodium Sahcylate, 10 to 2500 mM Potassium Acetate, 10 to 4000 mM Ammonium Acetate, 10 to 4000 mMLithium Acetate, 1 to 1000 mM Sodium Citrate, 10 to 4000 mM Sodium Chloride, 10 to 5000 mM Guanidine HCL, 0.01% to 5 % Charcoal, 0.01% to 5 % Tween-20, 0.01% to 6 % Triton-xlOO, 0.01% to 5 % PVP, 0.01% to 2 % PVPP, 0.01% to 1 % Sodium Azide, 1 to 1000 mM Sodium Hydrogen Phosphate, 1 to 500 mM Di Sodium Hydrogen Phosphate and 1 to 100 mM Ammonium Sulphate, 1 to 100 mM Potassium Phosphate, 0.01 to 25 % Chloroform, 0.01 to 25 % Hexane, 0.01 to 25 % Phenol.

[0025] Another embodiment of one of the several alternative compositions of CP Solution but not limited to O.lx to lOx concentrated buffer of the below can be used as follows: 0.01% to 3 % Lauramide DEA, 0.01% to 1 % Butylated Hydroxytoluene, 1 to 25 mM Tris, 1 to 30 mM EDTA, 0.1 to 6% SDS, 10 to 2500 mM Potassium Acetate,10 to 4000 mM Ammonium Acetate, 10 to 4000 mM Sodium Chloride, 0.01% to 2% Sodium Deoxycholate, 0.01% to 5 % Tween-20, 0.01% to 6 % Triton-xlOO, 10 to 5000 mM Guanidine HCL, 0.01% to 5 % SulfonatedCastor Oil, 0.01 to 25 % Chloroform, 0.1 % to 10% agar or its derivatives, Polyvinylpyrrolidone,Polyvinylpolypyrrolidone, 0.01% to 5 % PVP, 0.01% to 2 % PVPP, 0.01% to 1 % Sodium Azide.

[0026] CP Solution

[0024] and

[0025] along with Anionic Surfactants: Lithium dodecyl sulfate, Sodium octyl sulfate., Sodium pentanesulfonate, Sodium dodecyl sulfate, Sodium decyl sulfate, Sodium dodecylbenzenesulfonate, Sodium stearate, Magnesium stearate, Sodium allylsulfonate, Sulfonated castor oil, Sodium ethyl 2- sulfolaureate, Sodium diisobutyl sulfosuccinate, Dodecylbenzenesulfonic acid sodium salt, Sodium lignosulfonate, Sodium lauryl polyoxyethylene ether sulfate, Sodium nonylphenol polyoxyethylene ether sulfate, Sodium lauryl sulfate, Sodium oleyl sarcosinate, Sodium pyrrolidone carbonate, Sodium polyalkyl phenyl polyoxyethylene ether sulfate, Sec-alkyl sodium sulfate, Linear alkylbenzene sulfonates, Sodium n-octylsulfonate, Sodium poly[(naphthaleneformaldehyde)sulfonate], Sodium diamyl sulfosuccinate.Cationic Surfactants: Decyltrimethylammonium chloride,Cetyltrimethylethylammonium bromide, Dodecyl phenyl ammonium sulfate, Ammonium lauryl sulfate, Ammonium dodecylbenzenesulphonate.Nonionic Surfactants: Butylnaphthalenesulfonic acid sodium salt, Lignosulfonic acid, calcium salt, 1 -Dodecanesulfonic acid sodium salt, 3-(N,N- Dimethylpalmitylammonio) propanesulfonate, Disodium methylenebisnaphthalenesulfonate, Cleaner for heat-transfer oil furnace, LY 171883, Silk softener, Hydroxyaluminum distearate, Polyethylene, Propyleneglycol (beta- Naphthyl) (3-Sulfopropyl) Diether, Potassium Salt, 2 -Dodecylbenzenesulfonate, Dicyclohexyl sulfosuccinate sodium salt, Disodium 4-dodecyl-2,4'- oxydibenzenesulfonate, Organosilicon surfactant, Sulfonated ahphatic polyester, Sodium-N-methyl-N-oleyl taurate, Dihexyl sodium sulfo succinate, Dibasic leadstearate, Sodium methyl cocoyl taurate, Dodecyl triethanolamine sulfate, Manganous stearate, Calcium dodecylbenzene sulfonate, Disodium 4-[2-[(l-oxoundec-10- enyl) amino] ethyl] 2-sulfonatosuccinate, Fluorocarbon surfactant, Lamepon A, 1- Hexadecanesulfonic acid sodium salt, Surfactant, 1 -Pentanesulfonic acid sodium salt monohydrate, Cocamidopropyl betaine, Amidoaminosurfactants, Jiuma plate aminoacid surfactant, Cleaner LS, 2,6-Dimorphohn-4-ylpyrimidine-4-carboxylic acid, CA{12A} fatty alcohol polyoxyethylene ether ammonium sulfate, Stearyltoluene sodium sulfonate, Nonylphenyl polyoxyethylene ether sulfate triethanolamine, Sopa, Dispersing agent CNF, Sulfate AEC, Water-decreasing agent AF, Antistatic finish agent for synthetic fiber, Frothing agent K14, Glyceryl ethercarboxylic acid salt, Calcium stearyl lactate, Monoethanolamine dodecyl sulfate, Alkoxy ethanolamido sulfosuccinate sodium salt.

[0027] An embodiment of several alternative compositions of Precipitation Buffer but not limited to 0. lx to lOx concentrated buffer of the below can be used as follows: 1-2 M of Sodium Acetate, 1-5M Potassium Acetate, 1-3 M Ammonium Acetate, 1-4 M Lithium Acetate, 1-5 M Sodium Chloride, 1-2 M Potassium Chloride.

[0028] An embodiment of several alternative compositions of Binding Buffer but not limited to 0. lx to lOx concentrated buffer of the below can be used as follows: IM to 9 M Guanidinium Isothiocyanate and or IM to 9 M Guanidinium Hydrochloride, 0.1- 1 M of Sodium Citrate, 0.1-1 M of Phosphate Buffer, 1% to 20% Polyoxyethylene Ethers, Sodium Acetate, Sodium Chloride, Potassium Chloride, 1% to 50% tween 20, 1% to 20% Triton, 1% to 30%Sulfonated Castor Oil.

[0029] An embodiment of several alternative compositions of Wash Buffer but not limited to 0. lx to lOx concentrated buffer of the below can be used as follows: IM to 9 M Guanidinium Isothiocyanate and or IM to 9 M Guanidinium Hydrochloride, 0.1-1 M of Sodium Citrate, 0.1-1 M of Phosphate Buffer, Sodium Acetate, Sodium Chloride, Potassium Chloride, 1% to 50% Tween 20, 1% to 20% Triton, 1% to 30%Sulfonated Castor Oil.

[0030] An embodiment of several alternative compositions of Pre-Wash Buffer but not limited to O.lx to lOx concentrated buffer of the below can be used as follows: 1% to 70% Xylene, 10 to 70 % Butanol, 10 to 70% Methanol, 10 to 70% Isopropanol, 10 to 300 mM Phosphate Buffer.

[0031] Use of polymerases that are resistant to inhibitors along with CP solution and OM method.

[0032] Figure 1: Flowchart showing extraction of biomolecules like nucleic acids using CP Solution in OM Method and kits.

[0033] Cloudy Precipitated (CP) Solution for Extraction of All Biomolecules Including Nucleic Acids.

[0034] The Cloudy Precipitated (CP) solution remains cloudy and is not clear, even when heated with or without a sample.

[0035] Unlike other lysis buffers, the CP Solution lyse the cells and extracts nucleic acids and other biomolecules, even in the cloudy precipitated state.

[0036] Fig. 1 Part 1 - Empty Tube: Prepare an empty tube.

[0037] Fig. 1 Part 2 - CP Solution: Add the CP solution to the tube.

[0038] Fig. 1 Part 3 - Add Sample: Introduce the sample into the CP solution.

[0039] Fig. 1 Part 4 - Heat: Heat the mixture.

[0040] Fig. 1 Part 5 - Centrifuge: Centrifuge the heated mixture.

[0041] Fig. 1 Part 6 - Purified Biomolecules: Collect the purified biomolecules from the supernatant.

[0042] Figure 2: Flowchart showing choices of all different Embodiments used in the process of extraction of nucleic acids.

[0043] Based on the level of purity of nucleic acid in the one-minute method, the single CP Solution can be used alone in one step or can be further subjected to additional chemicals and steps for the desired purity.

[0044] Fig. 2 Part 7 - Sample: Collect the sample.

[0045] Fig. 2 Part 8 - Pre-treatment: Pre-treat the sample to prepare it for the extraction of nucleic acids.

[0046] Fig. 2 Part 9 - CP Solution: Add the CP solution.

[0047] Fig. 2 Part 10 - Tissue Homogenization, Heat, and Freeze: Homogenize the mixture, apply heat, and freeze.

[0048] Fig. 2 Part 11 - Centrifuge: Centrifuge to obtain the purified nucleic acids from the supernatant.

[0049] Fig. 2 Part 12 - Biological Applications: Utilize the extracted nucleic acids from the supernatant for various biological applications.

[0050] Fig. 2 Part 13 - Filter Membranes & Sephadex: Use filters membranes and Sephadex for further purification of nucleic acids from the supernatant.

[0051] Fig. 2 Part 14 - Biological Applications: Utilize the extracted nucleic acids for various biological applications.

[0052] Fig. 2 Part 15 - Precipitation: Use precipitation for further purification of nucleic acids from the supernatant.

[0053] Fig. 2 Part 16 - Biological Applications: Utilize the extracted nucleic acids for various biological applications.

[0054] Fig. 2 Part 17 - Magnetic sihca beads: Use Magnetic silica beads for further purification of nucleic acids from the supernatant.

[0055] Fig. 2 Part 18-Biological Applications: Utilize the extracted nucleic acids for various biological applications.

[0056] Fig. 2 Part 19 - Silica matrix: Use spin column for further purification of nucleic acids from the supernatant.

[0057] Fig. 2 Part 20 - Biological Applications: Utilize the extracted nucleic acids for various biological applications.

[0058] Fig. 2 Part 21 - Sihca powder (silica gel): Use silica powder (silica gel) for further purification of nucleic acids from the supernatant.

[0059] Fig. 2 Part 22 - Biological Applications: Utilize the extracted nucleic acids for various biological applications.

[0060] Fig. 3 - Steps - Embodiment 1

[0061] Fig. 3 Part 23 - Add the pre-treatment solution to the sample and vortex well. Centrifuge the mixture, then perform one or two pre-wash steps. Proceed with the following steps.

[0062] Fig. 3 Part 24 - Add the pre-washed sample to the CP Solution and vortex to mix well.

[0063] Fig. 3 Part 25 - Perform bead-beating or any suitable method for homogenization. Optionally, treat with proteinase K, RNAse, or DNAse, and heat inactivate for 10 minutes at 25 to 95°C, depending on the sample type, enzyme nature, and nucleic acid criteria. Incubate the mixture at 10°C to 95°C for a few minutes to a few hours. Keep the mixture at -20°C or on ice for a few minutes to a few hours.

[0064] Fig. 3 Part 26 - Centrifuge to remove impurities.

[0065] Fig. 3 Part 27 - Use the crude supernatant for biological applications. Store the supernatant containing nucleic acids at -20°C or -80°C.

[0066] Fig. 4 - Steps - Embodiment 2

[0067] Fig. 4 Part 28 - Add the pre-treatment solution to the sample and vortex well. Centrifuge the mixture, then perform one or two pre-wash steps. Proceed with the following steps.

[0068] Fig. 4 Part 29 - Add the pre-washed sample to the CP Solution and vortex to mix well.

[0069] Fig. 4 Part 30 - Perform bead-beating or any suitable method for homogenization. Optionally, treat with proteinase K, RNAse, or DNAse, and heat inactivate for 10 minutes at 25 to 95°C, depending on the sample type, enzyme nature, and nucleic acid criteria. Incubate the mixture at 10°C to 95°C for a few minutes to a few hours. Keep the mixture at -20°C or on ice for a few minutes to a few hours.

[0070] Fig. 4 Part 31 - Centrifuge to remove impurities.

[0071] Fig. 4 Part 32 - Pass the crude supernatant through filter membranes, Sephadex, a gel chromatography column with beads or resin, or any other absorbent material, such as charcoal, to remove salts and impurities. Collect the flow-through.

[0072] Fig. 4 Part 33 - The flow-through containing the nucleic acids can be used for biological applications. Store the supernatant containing nucleic acids at -20°C or -80°C.

[0073] Fig. 5 - Steps - Embodiment 3

[0074] Fig. 5 Part 34 - Add the pre-treatment solution to the sample and vortex well. Centrifuge the mixture, then perform one or two pre-wash steps. Proceed with the following steps.

[0075] Fig. 5 Part 35 - Add the pre-washed sample to the CP Solution and vortex to mix well.

[0076] Fig. 5 Part 36 - Perform bead-beating or any suitable method for homogenization. Optionally, treat with proteinase K, RNAse, or DNAse, and heat inactivate for 10 minutes at 25 to 95°C, depending on the sample type, enzyme nature, and nucleic acid criteria. Incubate the mixture at 10°C to 95°C for a few minutes to a few hours. Keep the mixture at -20°C or on ice for a few minutes to a few hours.

[0077] Fig. 5 Part 37 - Centrifuge to remove impurities.

[0078] Fig. 5 Part 38 - Add precipitation solution to the crude supernatant, mix well, freeze the mixture, and centrifuge. Discard the supernatant.

[0079] Fig. 5 Part 39 - Add Wash Buffer- 1 to the pellet, mix, and centrifuge. Discard the supernatant.

[0080] Fig. 5 Part 40 - Add Wash Buffer-2 to the pellet, mix, and centrifuge. Discard the supernatant and air dry the pellet.

[0081] Fig. 5 Part 41 - Add Elution Buffer to the pellet and mix well.

[0082] Fig. 5 Part 42 - Use the purified nucleic acids for biological applications. Store the supernatant containing nucleic acids at -20°C or -80°C.

[0083] Fig. 6 - Steps - Embodiment 4

[0084] Fig. 6 Part 43 - Add the pre-treatment solution to the sample and vortex well. Centrifuge the mixture and then perform one or two pre-wash steps. Proceed with the following steps.

[0085] Fig. 6 Part 44 - Add the pre-washed sample to the CP Solution and vortex to mix well.

[0086] Fig. 6 Part 45 - Perform bead-beating or any suitable method for homogenization. Optionally, treat with proteinase K, RNAse, or DNAse, and heat inactivate for 10 minutes at 25 to 95°C, depending on the sample type, enzyme nature, and nucleic acid criteria. Incubate the mixture at 10°C to 95°C for a fewminutes to a few hours. Keep the mixture at -20°C or on ice for a few minutes to a few hours.

[0087] Fig. 6 Part 46 - Centrifuge to remove impurities.

[0088] Fig. 6 Part 47 - Add binding solution and enhancer solution to the crude supernatant, mix well. Load the supernatant onto silica magnetic beads, mix well, and separate the magnetic beads using a magnetic separator. Discard the supernatant by pipetting.

[0089] Fig. 6 Part 48 - Add Wash Buffer- 1 to the silica magnetic beads, mix well, and separate the magnetic beads using a magnetic separator. Discard the supernatant by pipetting.

[0090] Fig. 6 Part 49 - Add Wash Buffer-2 to the silica magnetic beads, mix well, and separate the magnetic beads using a magnetic separator. Discard the supernatant by pipetting.

[0091] Fig. 6 Part 50 - Add Elution Buffer to the silica magnetic beads, incubate at room temperature or at 65°C for 5 to 10 minutes, and separate the magnetic beads using a magnetic separator. Pipette the eluted supernatant into a new tube.

[0092] Fig. 6 Part 51 - Use the purified nucleic acids for biological applications. Store the supernatant containing nucleic acids at -20°C or -80°C.

[0093] Fig. 7 - Steps - Embodiment 5

[0094] Fig. 7 Part 52 - Add the pre-treatment solution to the sample and vortex well. Centrifuge the mixture and then perform one or two pre-wash steps. Proceed with the following steps.

[0095] Fig. 7 Part 53 - Add the pre-washed sample to the CP Solution and vortex to mix well.

[0096] Fig. 7 Part 54 - Perform bead-beating or any suitable method for homogenization. Optionally, treat with proteinase K, RNAse, or DNAse, and heatinactivate for 10 minutes at 25 to 95°C, depending on the sample type, enzyme nature, and nucleic acid criteria. Incubate the mixture at 10°C to 95°C for a few minutes to a few hours. Keep the mixture at -20°C or on ice for a few minutes to a few hours.

[0097] Fig. 7 Part 55 - Centrifuge to remove impurities.

[0098] Fig. 7 Part 56 - Add binding solution and enhancer solution to the crude supernatant, mix well. Load the supernatant onto a silica matrix -based spin column and centrifuge. Discard the flow-through.

[0099] Fig. 7 Part 57 - Add Wash Buffer- 1 to the silica matrix-based spin column and centrifuge. Discard the flow-through.

[0100] Fig. 7 Part 58 - Add Wash Buffer-2 to the silica matrix-based spin column and centrifuge. Discard the flow-through. Air dry the silica matrix-based spin column.

[0101] Fig. 7 Part 59 - Add Elution Buffer to the sihca matrix-based spin column, incubate at room temperature or at 65°C for 5 to 10 minutes, and centrifuge. Save the flow-through.

[0102] Fig. 7 Part 60 - Use the purified nucleic acids for biological applications. Store the supernatant containing nucleic acids at -20°C or -80°C.

[0103] Fig. 8 - Steps - Embodiment 6

[0104] Fig. 8 Part 61 - Add the pre-treatment solution to the sample and vortex well. Centrifuge the mixture, then perform one or two pre-wash steps. Proceed with the following steps.

[0105] Fig. 8 Part 62 - Add the pre-washed sample to the CP Solution and vortex to mix well.

[0106] Fig. 8 Part 63 - Perform bead-beating or any suitable method for homogenization. Optionally, treat with proteinase K, RNAse, or DNAse, and heat inactivate for 10 minutes at 25 to 95°C, depending on the sample type, enzymenature, and nucleic acid criteria. Incubate the mixture at 10°C to 95°C for a few minutes to a few hours. Keep the mixture at -20°C or on ice for a few minutes to a few hours.

[0107] Fig. 8 Part 64 - Centrifuge to remove impurities.

[0108] Fig. 8 Part 65 - Add binding solution and enhancer solution to the crude supernatant, mix well. Load the supernatant onto silica powder (silica gel) and centrifuge. Discard the flow-through.

[0109] Fig. 8 Part 66 - Add Wash Buffer- 1 to the silica powder (silica gel) and centrifuge. Discard the flow-through.

[0110] Fig. 8 Part 67 - Add Wash Buffer-2 to the silica powder (silica gel) and centrifuge. Discard the flow-through. Dry heat the sihca powder (silica gel) at 65°C for 5 minutes.

[0111] Fig. 8 Part 68 - Add Elution Buffer to the silica powder (sihca gel), incubate at room temperature or at 65°C for 5 to 10 minutes, and centrifuge. Save the flow- through.

[0112] Fig. 8 Part 69 - Use the purified nucleic acids for biological applications. Store the supernatant containing nucleic acids at -20°C or -80°C.

[0113] Table 1: Extraction of Nucleic Acids from Blood Samples showing the performance of this invention in comparison to existing methods.

[0114] Embodiment- 1 and Embodiment-2 significantly outperformed the commercial kits in terms of Yield OD, CT Value, and Copy Number, showing up to 18-fold and 12-fold improvement, respectively.

[0115] Embodiment-3 also showed a high yield and copy number, but the commercial kits were inhibited, making direct comparison challenging.

[0116] Embodiment-5 showed slight improvement over the commercial kits in terms of copy number, though its yield was lower.

[0117] Table 2: PCR of Extracted Nucleic Acids from Stool Samples showing the performance of this invention in comparison to existing methods.

[0118] Embodiment- 1 and Embodiment-3 showed significantly lower CT values and higher yields, with Embodiment-3 achieving a notable 59-fold increase in copy number compared to the commercial kits.

[0119] Embodiment-2 and Embodiment-5 also showed improved performance, though the commercial kits were inhibited, making direct yield comparisons difficult.

[0120] Commercial kits consistently failed to produce results, indicated by inhibited values, making the embodiments significantly more effective for DNA extraction from stool samples.

[0121] Device for Biomolecule Extraction and Separation Using Integrated Thermal and Filtration Modules

[0122] Extraction Method for Plant Samples. The following methods outline different approaches for extracting nucleic acids and other biomolecules from plant samples, comparing traditional methods to the novel device and method proposed herein.

[0123] Single-Step Nucleic Acid and Biomolecule Extraction Tube. The single- step extraction tube simplifies the process by integrating heating and filtration into one device. This system efficiently lyses cells and extracts biomolecules, including nucleic acids, proteins, and peptides, in a single workflow. By incorporating multiple filter membranes within the same tube, the method reduces sample handling and contamination risks, leading to high yields and purity.

[0124] Double-Step Nucleic Acid and Biomolecule Extraction Tubes. The double-step extraction method divides the lysis and filtration processes into two steps, allowing for more targeted processing. The first tube focuses on cell lysis, and the second isolates the desired biomolecules. This method offers higher flexibility in processing complex samples, ensuring better isolation through sequential steps.

[0125] Commercial Kit Method. In contrast to the single- and double-step tubes, commercial kits generally involve multiple steps that include separate devices for lysis, binding, washing, and elution. These kits require manual transfer of the sample between steps, increasing contamination risk, reducing yield, and prolonging processing times. While effective, commercial kits often require more labor and materials, making them less efficient for high-throughput applications.

[0126] Traditional CTAB Method. The CTAB (cetyltrimethylammonium bromide) method is a widely used chemical-based approach for nucleic acid extraction. This method is labor-intensive and involves multiple steps, such as cell lysis using chemical reagents, phase separation, and nucleic acid precipitation. Although it provides decent nucleic acid yields, the process is time-consuming and prone to degradation, especially in samples with high contaminants or inhibitors.

[0127] Traditional Precipitation Method. Traditional precipitation methods use alcohols like ethanol or isopropanol to precipitate nucleic acids from the solution. Though cost-effective, this method often results in lower purity of extracted biomolecules as co-precipitation of contaminants occurs. Additionally, it lacks the automation and efficiency required for high-throughput applications.

[0128] Table 1: Performance Comparison of Extraction Methods for Plant Samples

[0129] DNA Measurement. DNA concentrations were measured using a Qubit 4 device with a high-sensitivity dsDNA reagent.

[0130] All DNA samples were diluted 10-fold prior to PCR analysis using universal 18S primers.

[0131] Extraction Method for Clinical Samples. The same novel extraction methods were tested on clinical samples to assess their versatility and efficiency compared to commercial kits and traditional methods.

[0132] Single-Step Nucleic Acid and Biomolecule Extraction Tube. The single-step tube method proved equally effective for clinical samples, providing high-quality nucleic acids in a single, streamlined process.

[0133] Double-Step Nucleic Acid and Biomolecule Extraction Tubes. Similar to the plant sample tests, the double-step method provided flexibility and improved biomolecule isolation for clinical samples.

[0134] Commercial Kit Method. Commercial kits, while commonly used in clinical settings, were outperformed by the novel method in terms of yield and time efficiency.

[0135] Traditional Precipitation Method. The traditional precipitation method was found to be the least efficient for clinical samples, with lower yield and more contamination.

[0136] Table 2: Performance Comparison of Extraction Methods for Clinical Samples.

[0137] Dilution for PCR. All DNA was diluted 10 times before being used in the PCR for consistent comparison across different methods.

[0138] Method and Device for Nucleic Acid Extraction

[0139] Device Description. The device described here is a system designed for efficient extraction of nucleic acids and other biomolecules, such as proteins and peptides, from biological samples. The method involves either a one-step or multiplestep process carried out within specialized tubes that integrate both sample preparation and filtration, minimizing the need for separate handling steps and reducing the risk of contamination.

[0140] Fig. 9: One- or Multiple-Step Extraction Using a Sample Tube. The sample tube plays a vital role in the process, serving not only as a container for the sample but also facilitating pre-filtration. This pre -filtration helps in removing impurities, such as cell debris, while preparing the sample for efficient biomolecule extraction.

[0141] Bed Volume Range. The volume of the sample tube ranges from 25 pL to 600 jiL, allowing flexibility in sample size for both small and large-volume extractions.

[0142] Fig. 9, Part 1 : End Cap. The end cap securely seals the sample tube, ensuring that no contaminants enter the system during the extraction process and helping to maintain a sterile environment.

[0143] Fig. 9, Part 2: End Closure. The end closure serves as a protective barrier and is critical for proper seahng during heating or centrifugation steps, ensuring pressure is maintained inside the tube for optimal biomolecule extraction.

[0144] Fig. 9, Part 3: Collection Tube Connecting Rib. This rib facihtates easy connection between the sample tube and the collection container. It ensures a smooth flow of extracted biomolecules into the collection tube, minimizing sample loss.

[0145] Fig. 9, Part 4: Packing Rings. The system is designed with 1 to 4 packing rings to ensure a tight fit between the tube sections. These rings help maintain the integrity of the device during high-speed centrifugation or pressure-based filtration, preventing leakage and ensuring efficient extraction.

[0146] Fig. 9, Part 5: Filter Membranes. The device integrates 1 to 10 filter membranes depending on the complexity of the sample. These membranes are critical in filtering out unwanted debris and small molecules, ensuring only the target biomolecules pass through. The different filter membranes have variable pore sizes, allowing for stepwise filtration of biomolecules of different molecular weights.

[0147] Fig. 9, Part 6: Membrane Packing Rib. The membrane packing rib holds the filter membranes in place. This ensures that the membranes do not shift during the extraction process, maintaining filtration efficiency and preventing crosscontamination between filtered substances.

[0148] Fig. 10: One or Multiple-Step Extraction Using a Filter Tube. The filter tube further enhances the separation of biomolecules by capturing and isolating molecules based on their size. The filters selectively capture nucleic acids or proteins while allowing smaller molecules to pass through, ensuring that only high-quality biomolecules are retained.

[0149] Bed Volume Range: The bed volume for the filter tube also ranges from 25 jiL to 600 pL, accommodating a wide range of sample types and volumes.

[0150] Fig. 10, Part 7: Connecting Point of Filter Tube. The connecting point ensures seamless integration with external collection containers, allowing for continuous flow of the extracted biomolecules into the downstream processing unit. This minimizes manual intervention, making the device suitable for automated systems.

[0151] Fig. 10, Part 8: Sephadex Filling. The tube contains 0.1 mL to 5 mL of Sephadex (1% to 5% concentration) as a gel filtration medium. Sephadex is useful in separating biomolecules based on size and helps ensure that smaller molecules, such as salts, are filtered out, while nucleic acids and proteins are retained.

[0152] Fig. 10, Part 9: Bead Packed Area (0.1 mm to 1 mm). This section is filled with beads of sizes ranging from 0.1 mm to 1 mm, which aid in the separation of biomolecules by size -exclusion chromatography. The beads allow smaller molecules to enter the pores, while larger biomolecules are excluded and collected for further analysis.

[0153] Fig. 10, Part 10: Bead Packed Area (1 mm to 5 mm). A second bead-packed area, with beads ranging from 1 mm to 5 mm, provides further refinement of the filtration process, enabling the isolation of larger biomolecules like intact nucleic acids and proteins.

[0154] Fig. 10, Part 11: Tube Cutting Area. The tube cutting area provides a defined location where the user can section off different parts of the tube for easy access to specific layers of the filter and the collected sample. This section is designed to allow the user to isolate different fractions without contaminating the entire sample.

[0155] Device Description for Nucleic Acid and Biomolecule Extraction Using Filtration Method. This device is designed for efficient extraction of nucleic acids and other biomolecules from biological samples, such as plant tissues or clinical samples. The method utihzes one- or multiple-step filtration, integrating sample lysis and biomolecule separation into a streamlined process. The device features a flexibledesign, accommodating different sample sizes, and includes options for both single - step and double-step extraction. The use of specific filters and membranes ensures precise isolation of nucleic acids, proteins, or peptides.

[0156] Fig. 11: Double-Step Extraction Using Two Devices. This method involves two separate devices for nucleic acid extraction. The first sample tube is used for lysis, and after lysis, the filter tube is attached to the sample tube for biomolecule separation.

[0157] Fig. 11, Part 12: Double-Step Nucleic Acid and Biomolecule Extraction Tube. This tube allows for sequential steps of lysis and filtration, ensuring a more thorough sep ar ation of biomolecules .

[0158] Fig. 12: Single-Step Extraction Using a Single Device. In this variation, the extraction is carried out using a single device where both the lysis and biomolecule separation happen in one tube. This simplifies the process, making it more suitable for quick extractions.

[0159] Fig. 12, Part 13: Single-Step Nucleic Acid and Biomolecule Extraction Tube. Combines lysis and filtration in one step, reducing the time and complexity compared to multi-step methods.

[0160] Fig. 13: Large-Size Sample Tube for High-Volume Extraction. This figure illustrates a larger sample tube designed to handle higher volumes, from 0.1 mL to 15 mL. This is particularly useful for bulk sample extractions.

[0161] Bed Volume Range. Between 0.1 mL to 15 mL, making it suitable for larger sample sizes.

[0162] Fig. 13, Part 14: Collection Tube (1.5 mL to 50 mL): Designed to accommodate a variety of sample volumes.

[0163] Fig. 13, Part 15: 1 to 10 Filter Membranes: Ensures effective filtration by using multiple membranes with varying pore sizes.

[0164] Fig. 13, Part 16: 1 to 4 Packing Rings: Maintains the integrity of the filtration process by tightly securing the membranes.

[0165] Fig. 14: Large-Size Filter Tube. This filter tube is designed to capture and isolate nucleic acids and other biomolecules from high-volume samples. It includes a combination of separation membranes, agarose, and Sephadex gel.

[0166] Bed Volume Range. Similar to the sample tube, it ranges from 0.1 mL to 15 mL.

[0167] Fig. 14, Part 17: Connecting Point of Filter Tube: Allows the filter tube to be securely attached to the sample tube for a continuous process.

[0168] Fig. 14, Part 18 and 19: Separation Membranes: Utilize 1 to 10 membranes with different properties to capture biomolecules of varying sizes.

[0169] Fig. 14, Part 20: Agarose: Helps in size exclusion, effectively separating nucleic acids from smaUer impurities.

[0170] Fig. 14, Part 21: Sephadex: Facilitates further purification, separating small molecules like salts from biomolecules.

[0171] Fig. 14, Parts 22 and 23: Bead Packed Area (0.1 mm to 5 mm): Enhances filtration by enabling size-exclusion chromatography.

[0172] Fig. 14, Part 24: Tube Cutting: Allows the tube to be sectioned at specific points for easy access to different layers of filtration.

[0173] Fig. 15: Double-Step Extraction for Large Samples. In this method, two devices are used for extraction. The first device handles the lysis of the sample, and the second device is used for filtration and biomolecule separation.

[0174] Fig. 15, Part 25: Double-Step Nucleic Acid and Biomolecule Extraction Tube. Optimized for large-scale sample extractions, ensuring thorough separation.

[0175] Fig. 16: Single-Step Extraction for Large Samples. This method uses a single large-sized device, where both lysis and filtration are performed in one tube.

[0176] Fig. 16, Part 26: Single-Step Nucleic Acid and Biomolecule Extraction Tube: Simplifies the process, making it ideal for high-throughput sample processing.

[0177] Fig. 17 and Fig. 10: Single-Step and Double-Step Nucleic Acid Extraction Device. The device is designed for nucleic acid extraction using either a single-tube or a two-step approach. In the single-step method, the sample is processed in one tube, followed by passing it through a splitter to separate components. The supernatant is then filtered through a Fine-Up filter tube or one or more membranes to isolate biomolecules. Filtration is based on molecular weight, effectively separating biomolecules such as nucleic acids, proteins, or peptides from smaller impurities.

[0178] Bed Volume Range: 0.1 mL to 15 mL, accommodating both small and large sample volumes.

[0179] Fig. 17, Part 27: 1 to 10 Filter Membranes: These membranes facilitate effective biomolecule separation based on molecular size, ensuring high-purity isolation.

[0180] Fig. 17, Part 28: Splitter: This component divides the sample flow, enabling different fractions to be processed individually.

[0181] Fig. 17, Part 29: Fine-Up Filter Tube: This tube provides precise filtration, ensuring the capture of smaller molecules and allowing for efficient biomolecule isolation.

[0182] Fig. 17, Part 30, 31 and 32: Sephadex and Bead Packed Areas: These components assist in the final stages of separating smaller contaminants from the desired biomolecules, improving purification results.

[0183] Fig. 17, Part 33: Tube Cutting: This allows for specific layers of the filter to be isolated, enabling targeted analysis or further processing.

[0184] Fig. 18, Part 34: Double-Step Nucleic Acid Extraction Device. This device is designed for nucleic acid extraction using either a single-tube or two-step approach. After initial extraction, the sample is passed through a sphtter to separatecomponents. The supernatant is then filtered through a Fine-Up filter tube or one or more membranes to isolate biomolecules. The filtration process is based on molecular weight, effectively separating biomolecules such as nucleic acids, proteins, or peptides from smaller impurities.

[0185] Fig. 19: Single-Step Extraction with Fine-Up Filter and Splitter. This figure illustrates the single-step extraction method, where a splitter and Fine-Up filter are used in conjunction to isolate biomolecules after sample lysis. The splitter plays a crucial role in separating components of the lysed sample, allowing only the supernatant which contains the target biomolecules to pass through the Fine-Up filter or one or more membranes. This controlled filtration ensures that biomolecule isolation is efficient, with separation based on molecular weight. The Fine-Up filter tube, integrated into this process, enables precise biomolecule isolation, resulting in the streamlined extraction of high-purity nucleic acids, proteins, or other biomolecules.

[0186] Fig. 19, Part 35: Single-Step Nucleic Acid and Biomolecule Extraction with Fine-Up Filter Tube. The single-step nucleic acid extraction system combines the splitter and Fine-Up filter tube to provide an optimized method for biomolecule extraction. The splitter ensures that only the relevant fractions of the sample such as the supernatant containing nucleic acids or proteins pass through the Fine-Up filter, effectively removing contaminants and enabling high -purity extraction. The filtration occurs in a single integrated step, where the Fine-Up filter selectively captures and isolates biomolecules based on their molecular size. This configuration significantly reduces processing time while maintaining accuracy and precision in the biomolecule separation process.

Claims

Claims1. A method for isolating nucleic acids from a biological sample using a One- to-Multiple (OM) Method in one or more processes, the method comprising:(a) contacting the biological sample with a cloudy precipitated (CP) solution, wherein the CP solution remains visibly cloudy and precipitated under processing conditions including heating and sample mixing;(b) wherein the CP solution comprises a buffer configured to induce cloudiness or precipitation; and(c) isolating the nucleic acids under the cloudy, precipitated conditions facilitated by the CP solution.

2. A method for preparing a composition of a CP solution, the method comprising formulating a buffer comprising one or more of the following components: 1 to 25 mM Tris, 1 to 30 mM EDTA, 1 to 50 mM EGTA, 0.01% to 5% CTAB, 0.1% to 6% SDS, 10 to 1000 mM sodium salicylate, 10 to 2500 mM potassium acetate, 10 to 4000 mM ammonium acetate, 10 to 4000 mM lithium acetate, 1 to 1000 mM sodium citrate, 10 to 4000 mM sodium chloride, 10 to 5000 mM guanidine HC1, 0.01% to 5% charcoal, 0.01% to 5% Tween-20, 0.01% to 6% Triton X-100, 0.01% to 5% PVP, 0.01% to 2% PVPP, 0.01% to 1% sodium azide, 1 to 1000 mM sodium hydrogen phosphate, 1 to 500 mM disodium hydrogen phosphate, 1 to 100 mM ammonium sulfate, 1 to 100 mM potassium phosphate, 0.01% to 25% chloroform, 0.01% to 25% hexane, and 0.01% to 25% phenol, wherein the composition comprises one or more of said components in selective concentrations.

3. A method for extracting nucleic acids and biomolecules from a sample, comprising heating the sample in a single-tube device, followed by filtering the sample through a filter tube or one or more membranes to isolate the nucleic acids and biomolecules based on molecular weight.

4. The method according to claim 1, wherein the CP solution is also used as a collection medium, storage medium, lysis buffer, and extraction buffer.

5. The method according to claim 4, wherein the CP solution contains powdered charcoal, agar, salts, detergents, and organic solvents in a cloudy, precipitated form.

6. The method according to any of claims 1 to 5, comprising detergents at 0.1% to 6% and salts at 10 to 2000 mM concentrations.

7. The method according to claim 6, further comprising chaotropic agents at 0.1 to 8 M and additional anionic detergents.

8. The method according to claim 2, wherein the CP solution includes stabilizers and surfactants at optimized concentrations.

9. The method according to claim 8, wherein the pH of the CP solution is between 4 and 9.

10. The method according to claim 9, wherein the CP solution is used at concentrations from 0.1X to 10X and diluted accordingly.

11. The method according to claim 10, wherein inhibitor-resistant polymerases are used with the CP solution and OM method.

12. The method according to claim 11, wherein the nucleic acids are purified using adsorbent matrix columns or filter tubes including multiple membranes with different pore sizes.

13. The method according to claim 12, wherein the prewash buffer includes alcohols and the precipitation / binding solutions include salts and chaotropic agents.

14. The method according to claim 2, wherein the CP solution includes carrier nucleic acids, either synthetic or isolated from biological materials.

15. The method according to claim 3, wherein the single-tube device includes an integrated heating and filtration system.

16. The method according to claim 3, wherein the sample tube volume ranges from 25 jrL to 25 mL.

17. The method according to claim 3, wherein the filter tube accommodates sequential filtration based on biomolecule size.

18. The method according to claim 3, wherein the single-tube device is designed for minimal sample handling and is automation-compatible.

19. The method according to claim 3, wherein the biomolecules include nucleic acids, proteins, peptides, and other biological macromolecules.

20. The method according to claim 3, wherein the lysis tube is followed by a filter tube with multiple filter membranes for stepwise filtration of biomolecules.

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