Method and articles for processing polynucleic acids from plasma

Abstract

A system for isolating and purifying low abundance, low molecular weight cell-free DNA (cfDNA) from blood plasma is provided. The system comprises a surface-modified solid support with functionalized chemical groups, such as polymerizable carboxylic acids for binding and recovery of cfDNA while minimizing interference from higher molecular weight genomic DNA. The method involves passing the plasma sample through the functionalized support, and selectively binding cfDNA to the support surface. Elution is achieved at a high pH, facilitating efficient cfDNA release. The invention further includes devices, such as pipet tips and columns with integrated functionalized supports, and a kit containing all necessary components for extraction and purification of low abundance, low molecular weight cfDNA from higher molecular weight genomic DNA.

Description

[0001] PA102159W002

[0002] METHOD AND ARTICLES FOR PROCESSING POLYNUCLEIC ACIDS FROM PLASMA

[0003] Technical Field

[0004] The present invention relates to methods for processing and isolating cell-free polynucleic acids, specifically low-abundance cell-free DNA (cfDNA) from blood plasma. The invention further includes surface-modified solid supports, devices, and kits to allow the method.

[0005] Background

[0006] The ability to isolate circulating cell-free DNA (cfDNA) such as low-molecular-weight fragments shed from tumor cells, has the potential to transform diagnostics and disease monitoring, particularly in oncology. Unlike traditional solid tissue biopsies, liquid biopsy via cfDNA extraction from blood plasma is minimally invasive and provides a more comprehensive, real-time assessment of a patient's disease state. However, cfDNA is present in blood plasma at extremely low concentrations, and current methods struggle to efficiently isolate cfDNA without contamination by higher molecular weight genomic DNA fragments released from blood cells. This contamination reduces cfDNA recovery rates, hinders accurate sequencing, and increases the risk of false-negative results, especially in applications requiring high sensitivity, such as early cancer detection or monitoring minimal residual disease.

[0007] Typical cfDNA isolation involves drawing blood and processing the plasma, yet conventional extraction technologies rely on techniques developed for general nucleic acid extraction, such as using silica membranes or magnetic beads, which are not optimized for the specific properties and low abundance of cfDNA. These methods often require the use of high salt concentrations and chaotropic agents, leading to issues with yield, purity, and increased processing steps. Additionally, the physical properties of cfDNA, such as its smaller size and unique surface chemistry, demand tailored isolation methods to enhance binding specificity and elution efficiency. Increasing the volume of blood collected to overcome these limitations is impractical and unsustainable, particularly in high-throughput or cost-sensitive settings.

[0008] Therefore, there is a growing need for innovative methods and materials that can selectively bind and elute cfDNA, particularly low abundance, low molecular weight cfDNA while minimizing genomic DNA contamination, allowing for higher purity and yield.

[0009] Summary

[0010] Accordingly, the present invention addresses the issues associated with efficient isolation of cell-free DNA (cfDNA) from blood plasma by providing a specialized method and materials for selective cfDNA binding and separation. These aspects address challenges associated with low abundance, low molecular weight cfDNA and its isolation from higher molecular weight genomic DNA.In one aspect, the invention features a surface-modified solid support comprising a nonwoven substrate grafted with polymerizable carboxylic acid groups. This functionalized substrate allows selective binding of cfDNA, enhancing the isolation process while reducing interference from higher molecular weight genomic DNA.

[0011] In another aspect, the invention includes a process for cfDNA isolation that involves plasma dilution and the addition of specific reagents, such as non-ionic surfactants and buffering agents, which improve the binding efficiency of cfDNA to the nonwoven support. Following binding, cfDNA is eluted at a high pH, providing a streamlined method for obtaining high-purity cfDNA.

[0012] A further aspect of the invention involves a secondary purification process using magnetic beads functionalized with a complexing agent and an amine compound. This magnetic bead-based method effectively separates low abundance, low-molecular-weight cfDNA from higher molecular weight genomic DNA, enabling a more refined and high-yield isolation.

[0013] In yet another aspect, the invention encompasses kits that integrate the functionalized nonwoven support and magnetic beads, along with necessary reagents and buffering agents, into a cohesive system. These kits are designed for ease of use with potential configurations in pipet tips, filtration units, and other devices that facilitate the binding, washing, and elution processes.

[0014] These aspects collectively provide an efficient, scalable solution for the isolation of low abundance, low molecular weight cfDNA from higher molecular weight genomic DNA and addresses the limitations of conventional methods and enabling a higher standard of purity and yield essential for diagnostic and therapeutic applications.

[0015] The details of one or more examples of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

[0016] Brief Description of Drawings

[0017] Exemplary embodiments disclosed herein may be more completely understood in consideration of the following detailed description in connection with the following figures. The figures are not necessarily drawn to scale. Like numbers used in the figures refer to like components. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number.

[0018] FIG. 1 illustrates the recovery efficiency of 334 base pair DNA fragments from plasma using mixed l,7,10,16-tetraoxa-4,13-diazacyclooctadecane (KRYPTOFIX 22 orK22). It highlights a combination of 90 mol% K22 and 10 mol% Morpholine to achieve the highest DNA recovery.

[0019] FIG. 2 illustrates the binding and recovery of 167 bp DNA fragments from a 40% plasma solutionusing various nonwoven formulations, with and without K22 modification, utilizing high-pH conditions to enhance DNA recovery.

[0020] Detailed Description

[0021] The present invention relates to methods for processing and isolating cell-free polynucleic acids, particularly low-abundance, low-molecular-weight cell-free DNA (cfDNA) from blood plasma. This invention further encompasses surface-modified nonwoven supports, integrated devices, and kits designed to enhance the efficiency and yield of the isolation process.

[0022] In the following description, reference is made to the accompanying figures that form a part thereof and in which various embodiments are shown by way of illustration. It is to be understood that other embodiments are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense.

[0023] In the following disclosure, the following definitions are adopted.

[0024] As used herein, all numbers should be considered modified by the term “about”. As used herein, “a,” “an,” “the,” “at least one,” and “one or more” are used interchangeably.

[0025] The words "preferred" and "preferably" refer to embodiments described herein that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0026] Reference throughout this specification to "one embodiment," "certain embodiments," "one or more embodiments" or "an embodiment" means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases such as "in one or more embodiments," "in certain embodiments," "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0027] The term cfDNA herein implies cell-free DNA. It also includes low abundance, low molecular weight cfDNA. It may be single-stranded nucleic acids comprising at least 50 bases, 100 bases, 160 bases, 200 bases, or 500 bases. Alternatively, it may be double-stranded nucleic acids, particularly comprising lengths of at least 50 base pairs (bp), 100 bp, 160 bp, 200 bp, or 500 bp.

[0028] The present invention provides a system for isolation of cfDNA, particularly low abundance, low molecular weight cfDNA from higher molecular weight genomic DNA.It comprises a surface-modified nonwoven support for initial cfDNA binding, followed by a secondary purification step using functionalized magnetic beads. Together, these components improve cfDNA recovery rates, reduce genomic DNA contamination, and streamline the workflow, making the process suitable for both clinical and laboratory environments.

[0029] In one embodiment, it comprises a surface-modified nonwoven support for selectively binding cfDNA from blood plasma. The nonwoven support herein is grafted with polymerizable carboxylic acid groups, which enhances cfDNA binding affinity while reducing non-specific binding of higher molecular weight genomic DNA. The nonwoven support may be made up of a polymeric material.

[0030] The polymeric material may include polypropylene, polyethylene, polyester, nylon, polyvinyl chloride, cellulose-based fibres, or combinations thereof.

[0031] The polymerizable carboxylic acid may be selected from the group consisting of methacrylic acid (MAA), acrylic acid, itaconic acid, maleic acid, or combinations thereof.

[0032] By incorporating carboxylic acid groups, the nonwoven support provides an effective substrate for isolating cfDNA, particularly low-molecular-weight fragments.

[0033] In a preferred embodiment, polymerizable carboxylic acid is methacrylic acid (MAA).

[0034] The proportion of polymerizable carboxylic acid groups grafted onto the substrate may be varied to optimize binding. For example, the mass gain of the substrate upon grafting may vary from about 20% to about 300%. The proportion of MAA in the grafted polymer may vary from about 20% to 100%, depending upon comonomer incorporation as described below.

[0035] Further, the surface-modified solid support described herein above may optionally be functionalized with monomers selected from glycidyl methacrylate (GMA) and N-vinyl-2-pyrrolidone (NVP) to modify the hydrophilicity of the support or to enhance the binding properties for cell-free DNA (cfDNA) isolation.

[0036] Functionalization with GMA introduces epoxy groups onto the surface of the nonwoven support, which, under the aqueous conditions of the grafting reaction, are believed to be hydrolyzed to diol groups, generating hydrophilic character to the support. In addition, functionalization with NVP adds hydrophilic properties to the nonwoven support, promoting enhanced sample flow and increasing the surface interaction between cfDNA and the support.

[0037] Surface-modified solid support comprising a nonwoven support grafted with polymerizable carboxylic acid may be rigid. Alternatively, the nonwoven support comprising a nonwoven support grafted with polymerizable carboxylic acid may be flexible.

[0038] In an embodiment, surface-modified solid support comprising a nonwoven support is configured and integrated for use in a device selected from the group consisting of a pipet tip, a column, and / or a filtration unit. This configuration allows for the binding, washing, and elution of cell-free polynucleic acidswithin a single device, making the process efficient and suitable for high-throughput laboratory settings. A method for processing cell-free polynucleic acids, specifically focusing on the isolation of cfDNA from plasma using a surface-modified nonwoven support is further provided. It involves sequential steps designed to enhance cfDNA binding, separation of low-abundance, low-molecular-weight cfDNA from higher molecular weight genomic DNA, and efficient elution under specific conditions.

[0039] The method begins by diluting a plasma sample with a non-ionic surfactant and a buffering agent. The addition of these agents serves to reduce protein interference, enhance cfDNA binding affinity, and stabilize pH conditions favorable for cfDNA isolation.

[0040] The non-ionic surfactant may be selected from polyoxyethylene-based surfactants such as polysorbates, alkylphenol ethoxylates, alkyl polyglucosides, and polyoxyethylene glycol alkyl ethers, or combinations thereof.

[0041] In an embodiment, non-ionic surfactant used is polyoxyethylene (20) cetyl ether.

[0042] The buffering agent may be selected from sodium acetate, phosphate buffers, citrate buffers, bicarbonate buffers, carbonate-bicarbonate, glycine-sodium hydroxide, borate, or tris(hydroxymethyl)aminomethane (TRIS) buffer.

[0043] After sample preparation, the plasma is passed through a nonwoven support grafted with polymerizable carboxylic acid groups.

[0044] The nonwoven support is constructed from a polymeric material such as polypropylene, polyethylene, polyester, nylon, polyvinyl chloride, or cellulose-based fibers, or combinations thereof.

[0045] Polymerizable carboxylic acid groups, selected from methacrylic acid (MAA), acrylic acid, itaconic acid, maleic acid, or combinations thereof, facilitate cfDNA binding through electrostatic and hydrogen-bond interactions.

[0046] In a preferred embodiment, polymerizable carboxylic acid is methacrylic acid (MAA).

[0047] Optionally, the nonwoven support may be further functionalized with monomers like glycidyl methacrylate (GMA) and N-vinyl-2 -pyrrolidone (NVP) to increase hydrophilicity and improve sample flow dynamics.

[0048] Following the binding step, cell-free poly nucleic acids are eluted from the nonwoven support by adjusting the pH to around 10 using a buffering agent combined with a non-ionic surfactant. This pH adjustment disrupts the electrostatic and hydrogen bonds, allowing cfDNA to be released from the support with high efficiency.

[0049] In an embodiment, the method further includes a bind and elute procedure utilizing magnetic beads that are functionalized with a complexing agent and an additional amine compound in a specified molar ratio. This procedure serves as a secondary purification step, where the complexing agent enhances cfDNA selectivity.Complexing agent is selected from the group consisting of synthetic cyclic polyether amine, a heterocyclic aliphatic group selected from fluorinated amine, piperazine or derivatives thereof, comprising N-methyl piperazine, N-phenyl piperazine, N-(2 -hydroxy ethyl) piperazine, N-(4-methoxyphenyl) piperazine, N-(4-trifluoromethylphenyl) piperazine, and l-(4-bromophenyl) piperazine.

[0050] In an embodiment, the complexing agent is synthetic cyclic polyether amine, preferably KRYPTOFIX 22.

[0051] The additional amine compound, facilitates to adjust the binding properties of the cfDNA.

[0052] In a preferred embodiment, an additional amine compound is morpholine.

[0053] The complexing agent and amine compound are present in a molar ratio of about 10:90 to 90:10, optimizing cfDNA separation.

[0054] To facilitate a single streamlined workflow, the nonwoven support may be integrated into devices such as pipet tips, columns, or filtration units. This configmation allows for the binding, washing, and elution of cell-free polynucleic acids within a single device, making the process efficient and suitable for high-throughput laboratory settings.

[0055] In yet another embodiment, the present invention provides a kit for the purpose of extraction and purification of cell-free polynucleic acids from blood plasma. It comprises functionalized nonwoven support made from a polymeric material selected from the group consisting of polypropylene, polyethylene, polyester, nylon, polyvinyl chloride, cellulose -based fibres, or combinations thereof., grafted with polymerizable carboxylic acids selected from methacrylic acid (MAA), acrylic acid, itaconic acid, maleic acid; or combinations thereof;

[0056] reagent mixture comprising a polyoxyethylene-based non-ionic surfactant selected from the group consisting of polysorbates, alkylphenol ethoxylates, alkyl polyglucosides, polyoxyethylene glycol alkyl ethers, or combinations thereof;

[0057] and a buffering agent selected from the group consisting of sodium acetate, phosphate buffers, citrate buffers, bicarbonate buffers, carbonate-bicarbonate, glycine-sodium hydroxide, borate, and tris(hydroxymethyl)aminomethane (TRIS) buffer.

[0058] In an embodiment, the nonwoven support may be optionally functionalized with monomers selected from glycidyl methacrylate (GMA) and n-vinyl-2-pyrrolidone (NVP) to enhance the binding capacity and specificity for cell-free polynucleic acids.

[0059] The nonwoven support may be integrated into a device, such as a pipet tip, column, or filtration unit, allowing for a streamlined, single-step workflow for binding, washing, and elution of cfDNA. This configmation enables easy handling, minimizes sample loss, and supports rapid, high-throughput processing of multiple samples, which is advantageous for laboratory settings requiring efficient cfDNA isolation.In embodiments, the kit can further comprise magnetic beads functionalized with complexing agent and an additional amine compound, in a specific ratio for separation of cell-free polynucleic acids from higher molecular weight genomic DNA.

[0060] The complexing agent may be selected from the group consisting of synthetic cyclic polyether amine, a heterocyclic aliphatic group selected from fluorinated amine, piperazine or derivatives thereof, comprising N-methyl piperazine, N-phenyl piperazine, N-(2-hydroxyethyl) piperazine, N-(4-methoxyphenyl) piperazine, N-(4-trifluoromethylphenyl) piperazine, and l-(4-bromophenyl) piperazine.

[0061] In an embodiment, the complexing agent is synthetic cyclic polyether amine, preferably KRYPTOFIX 22.

[0062] In a further embodiment, an additional amine compound is morpholine.

[0063] In a still further embodiment, the complexing agent and amine compound are present in a molar ratio between 10:90 to 90:10.

[0064] Additionally, the kit includes calibration standards to quantify the yield and purity of the isolated cfDNA.

[0065] Further, to facilitate ease of use, the kit is accompanied by detailed instructional materials. These instructions provide a step-by-step protocol for optimal use of the kit, covering all aspects of the process, including sample preparation, cfDNA binding, washing, and elution. The instructional materials are designed with clear guidelines on reagent handling, recommended incubation times, optimal pH ranges, and buffer preparation, ensuring that each step is executed effectively. Additionally, troubleshooting tips may be also provided to help users in separating the low abundance, low molecular weight cfDNA from higher molecular weight genomic DNA.

[0066] Unless otherwise indicated, all numbers expressing feature sizes, amounts, and physical properties used in the specification and claims are to be understood as being modified by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein.

[0067] Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a variety of alternate and / or equivalent implementations can be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific embodiments discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof.

[0068] EXAMPLES:Example 1: Preparation of Functionalized Nonwoven Support for Cell-Free DNA (cfDNA) Binding A nonfunctionalized melt-blown polypropylene microfiber nonwoven web having an effective fiber diameter (EFD) of 6 micrometres, was grafted with a nitrogen purged grafting solution. A 5 inch x 7 inch sheet sample of nonwoven web was cut, weighed, and inserted into 10 inch x 13 inch (25.4 cm x 33.0 cm) polyethylene bag having zip-lock type closures. A grafting solution was prepared in deionized water comprising, by weight, methacrylic acid (5%), NVP (12%), and GMA (4%). The solution was sparged with nitrogen for 2 minutes to remove dissolved oxygen from the solution. Sparging was accomplished by fully immersing a nitrogen line in the solution and bubbling nitrogen through the solution. Nonwoven sheet samples in bags and bottles of grafting solutions were transferred into a nitrogen purged glove box with an oxygen content of less than 20 ppm. Bags and grafting solution bottles were opened in the nitrogen purged glove box to purge the headspaces of the bags and bottles. Sample bags were then sealed and removed from the glove box. The sealed bags were taped to a carrier web being conveyed at 35 fpm (feet per minute) through an Energy Sciences, Inc (Wilmington, MA) ElectroCure electron beam. Samples were irradiated at 300 kV to a dose of 10 Mrad. Sealed sample bags were immediately returned to the glove box and saturated with a grafting solution by pouring the grafting solution into the bag. A roller was used to facilitate saturation of the nonwoven with the solution. The bag was resealed and maintained in the nitrogen atmosphere of the glove box for 3 hours. Next, the bag was removed from the glove box and opened in a fume hood to allow atmospheric oxygen into the bags. The functionalized nonwoven (FNW) sheet was then washed by immersing the sheet in a pot containing boiling deionized water for 90 minutes. The functionalized nonwoven sheet was then dried at ambient conditions in a polyethylene-lined aluminium tray. After ambient drying, the FNW sheet was weighed and placed in a 70°C convection oven for 30 minutes, after which it was weighed again. The mass difference was calculated as a percent of the final mass from the previous weight measurement of the FNW sheet. If the percent mass difference was more than 2%, the FNW sheet was returned to the oven, in 30-minute increments, until the percent mass difference from the previous weight measurement was less than 2%. The percent weight gain of the nonwoven substrate following the grafting and drying procedure was 223%.

[0069] Additional ftmctionalized nonwoven samples were prepared using aqueous grafting solutions comprising MAA alone, MAA plus NVP (various ratios), or MAA, NVP, and GMA (various ratios).

[0070] Example 2: Isolation of cfDNA from Plasma Using Functionalized Nonwoven Support A frozen plasma sample is thawed in a water bath at 37°C for 15 minutes. The plasma sample is then diluted in a ratio of 1:1.5 with deionized water and polyoxyethylene (20) cetyl ether (a non-ionic surfactant) is added at a final concentration of 1% and sodium acetate solution (pH 4.5) is added to a final concentration of 100 mM, to improve cfDNA binding. The nonwoven support prepared in Example 1 is washed with 20mM Tris-HCl pH 7.5, then equilibrated with 30 mM sodium acetate pH4.5. The dilutedplasma sample is passed through the functionalized nonwoven support at a flow rate of 10 ml / min, allowing cfDNA to bind to the carboxylated surface. Bound cfDNA is eluted from the nonwoven support using an elution buffer adjusted to pH 10, to release the cfDNA into solution. The eluted cfDNA solution is collected for processing with magnetic beads as described in Example 3 below.

[0071] Example 3: Enhanced cfDNA Purification Using Magnetic Beads with Complexing Agents cfDNA from plasma is bound and eluted from the functionalized nonwoven support as described in Example 2. Magnetic beads functionalized with a complexing agent, such as KRYPTOFIX 22, and an additional amine compound, such as morpholine, are equilibrated in prepared in 10 mM sodium acetate pH 4.5 buffer solution. 100 ul of IM Sodium acetate pH 4.5, 100 ul of 1% polyoxyethylene surfactant, and 10 ul of equilibrated magnetic beads (10 mg / ml) are added to 800 ul of nonwoven eluate. The cfDNA eluted from the nonwoven support is then incubated with the magnetic beads for 10 minutes at room temperature to allow further binding. The beads with bound cfDNA are washed with a buffer containing 50 mM Tris, 100 mM NaCl, and 0.1% polyoxyethylene surfactant to remove any residual contaminants. Finally, cfDNA is eluted from the magnetic beads with an elution buffer at pH 8.5, yielding high-purity cfDNA free from larger genomic DNA contaminants.

[0072] Example 4: Reduction in Charge Density on Bead Surface for Enhanced cfDNA Binding This example demonstrates the enhancement of cfDNA binding and recovery from plasma by decreasing the charge density on the bead surface. By testing various molar ratios of 1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (KRYPTOFIX 22 or K22) and morpholine, the effect on cfDNA binding efficiency was evaluated. Formulation was tested for its ability to recover a 334 base pair DNA fragment from bovine plasma. The ratios of K22 to Morpholine ranged from 100 mol% K22 alone to 100 mol% Morpholine alone, allowing assessment of both single-component and mixed-component surface modifications. The recovery efficiency of cfDNA for each formulation was recorded. The results showed that a 90 mol% K22 and 10 mol% Morpholine mixture achieved the highest cfDNA recovery, approximately 60%. This combination outperformed other ratios, where increasing Morpholine content beyond 10 mol% or using 100 mol% K22 or Morpholine alone resulted in significantly lower recovery rates. Results are shown in Fig 1.

[0073] For optimal surface charge for cfDNA binding, zeta potential (Zp) measurements were taken under two pH conditions: 10 mM NaOAc at pH 4.5 and 10 mM Tris HO at pH 8.5. The 90 mol% K22 and 10 mol% Morpholine mixture exhibited Zp values of -7 mV at pH 4.5 and -32 mV at pH 8.5, reflecting a balanced surface charge ideal for selective cfDNA binding while minimizing non-specific interactions. Results are shown in table 1 below:Table 1: Zeta Potential Measurements and Surface Charge Optimization

[0074]

[0075] Example 5: Evaluation of effectiveness of nonwoven support and Optimization of Functionalized Polypropylene Nonwovens for DNA Binding and Elution

[0076] Polypropylene nonwovens were prepared with varying graft densities of methacrylic acid (MAA), with and without glycidyl methacrylate (GMA), to evaluate their effectiveness in binding and eluting a 167 bp DNA fragment from plasma. Some nonwovens were also coupled with KRYPTOFIX-22 (K22), a poly ether amine, to assess its influence on DNAbinding and elution efficiency. FIG.2 illustrates the binding and recovery of 167 bp DNA fragments from a 40% plasma solution using these nonwoven formulations, showing the percentage of DNA bound and recovered in the eluate. Samples modified with K22 displayed varying binding and recovery levels, while samples without K22 exhibited comparable or slightly improved binding efficiency. Results emphasized the importance of pH 10 in enhancing DNA elution, as the higher pH likely disrupts interactions between DNA and the nonwoven matrix, particularly in K22-coupled samples where elution was otherwise challenging.

[0077] Unless otherwise indicated, all numbers expressing feature sizes, amounts, and physical properties used in the specification and claims are to be understood as being modified by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein.

[0078] Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a variety of alternate and / or equivalent implementations can be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific embodiments discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof.

Claims

CLAIMS1. A surface-modified solid support for binding cell-free polynucleic acids from blood plasma, comprising a nonwoven support grafted with polymerizable carboxylic acid groups.

2. The surface-modified solid support of claim 1, wherein the cell-free polynucleic acids are low abundance, low molecular weight cell-free DNA (cfDNA).

3. The surface-modified solid support of claim 1, wherein the cell-free poly nucleic acids are separated from higher molecular weight genomic DNA.

4. The surface-modified solid support of claim 1, wherein the polymerizable carboxylic acid is selected from the group consisting of methacrylic acid (MAA), acrylic acid, itaconic acid, maleic acid, or combinations thereof.

5. The surface-modified solid support of claim 1 , wherein the polymerizable carboxylic acid is methacrylic acid (MAA).

6. The surface-modified solid support of claim 1, wherein the nonwoven support is made from a polymeric material selected from the group consisting of polypropylene, polyethylene, polyester, nylon, polyvinyl chloride, cellulose-based fibres, or combinations thereof.

7. The surface-modified solid support of claim 1, wherein the nonwoven support is optionally functionalized with monomers selected from glycidyl methacrylate (GMA) and N-vinyl-2 -pyrrolidone (NVP).

8. The surface-modified solid support of claim 1, wherein the nonwoven support is configured for use in a device selected from the group consisting of a pipet tip, a column, a filtration unit, and combinations of two or more thereof.

9. A method for processing cell-free polynucleic acids from plasma using a surface-modified solid support, comprising:diluting a plasma sample and adding a non-ionic surfactant and a buffering agent to form a prepared sample;passing the prepared sample through a nonwoven support grafted with polymerizable carboxylic acid;eluting the cell-free polynucleic acids from the nonwoven support using a non-ionic surfactant agent and buffering agent at a pH of 10; andseparating the cell-free polynucleic acids from higher molecular weight genomic DNA.

10. The method of claim 9, further comprising performing a bind and elute procedure using magnetic beads functionalized with a complexing agent and an amine compound in a molar ratio, and adjusting the pH as necessary to facilitate binding and elution of the cell-free polynucleic acids.

11. The method of claim 9, wherein the cell-free poly nucleic acids are low abundance, low molecular weight cell-free DNA (cfDNA).

12. The method of claim 9, wherein the polymerizable carboxylic acid is selected from the group consisting of methacrylic acid (MAA), acrylic acid, itaconic acid, maleic acid, or combinations thereof.

13. The method of claim 9, wherein the polymerizable carboxylic acid is methacrylic acid (MAA).

14. The method of claim 9, wherein the nonwoven support is made from a polymeric material selected from the group consisting of polypropylene, polyethylene, polyester, nylon, polyvinyl chloride, cellulose- based fibres, or combinations thereof.

15. The method of claim 9, wherein the nonwoven support is optionally functionalized with monomers selected from glycidyl methacrylate (GMA) and N-vinyl-2 -pyrrolidone (NVP).

16. The method of claim 9, wherein the non-ionic surfactant is a polyoxyethylene-based non-ionic surfactant selected from the group consisting of polysorbates, alkylphenol ethoxylates, alkyl polyglucosides, polyoxyethylene glycol alkyl ethers, or combinations thereof.

17. The method of claim 9, wherein the non-ionic surfactant is polyoxyethylene (20) cetyl ether.

18. The method of claim 9, wherein the buffering agent is selected from the group consisting of sodium acetate, phosphate buffers, citrate buffers, bicarbonate buffers, carbonate-bicarbonate, glycine-sodium hydroxide, borate, and tris(hydroxymethyl)aminomethane (TRIS) buffer.

19. The method of claim 10, wherein the complexing agent is selected from the group consisting of (i) synthetic cyclic polyether amine, (ii) a piperazine or derivative thereof selected from N-methyl piperazine, N-phenyl piperazine, N-(2-hydroxyethyl) piperazine, N-(4-methoxyphenyl) piperazine, N- (4-trifluoromethylphenyl) piperazine, and l-(4-bromophenyl) piperazine, and (iii) a fluorinated amine.

20. The method of claim 10, wherein the complexing agent is synthetic cyclic polyether amine.

21. The method of claim 10, wherein the complexing agent is l,7,10,16-tetraoxa-4,13- diazacyclooctadecane.

22. The method of claim 10, wherein the amine compound is morpholine.

23. The method of claim 10, wherein the complexing agent and additional amine are present in a molar ratio between 10:90 to 90:10.

24. The method of claim 9, wherein the nonwoven support is integrated into a device selected from a pipet tip, a column, or a filtration unit, facilitating binding, washing, and elution of cell -free poly nucleic acids in a single workflow.

25. A kit for extraction and purification of cell-free polynucleic acids from blood plasma, comprising:a functionalized nonwoven support made from a polymeric material selected from the group consisting of polypropylene, polyethylene, polyester, nylon, polyvinyl chloride, cellulose -based fibres, or combinations thereof, and grafted with polymerizable carboxylic acids selected from methacrylic acid (MAA), acrylic acid, itaconic acid, maleic acid; or combinations thereof;reagent mixture comprising a polyoxyethylene-based non-ionic surfactant selected from the group consisting of polysorbates, alkylphenol ethoxylates, alkyl poly glucosides, polyoxyethylene glycol alkyl ethers, or combinations thereof;and a buffering agent selected from the group consisting of sodium acetate, phosphate buffers, citrate buffers, bicarbonate buffers, carbonate-bicarbonate, glycine-sodium hydroxide, borate, and tris(hydroxymethyl)aminomethane (TRIS) buffer; andmagnetic beads functionalized with complexing agent and an amine compound, in a molar ratio for separation of cell-free polynucleic acids from higher molecular weight genomic DNA.

26. The kit of claim 25, wherein the complexing agent and amine compound are present in a ratio between 10:90 to 90:10.

27. The kit of claim 26, wherein the complexing agent is selected from the group consisting of (i) synthetic cyclic polyether amine, (ii) a piperazine or derivative thereof selected from N-methyl piperazine, biphenyl piperazine, N-(2-hydroxyethyl) piperazine, N-(4-methoxyphenyl) piperazine, N-(4- trifluoromethylphenyl) piperazine, and l-(4-bromophenyl) piperazine, and (iii) a fluorinated amine.

28. The kit of claim 25, wherein the complexing agent is synthetic cyclic polyether amine.

29. The kit of claim 25, wherein the complexing agent is l,7,10,16-tetraoxa-4,13-diazacyclooctadecane.

30. The kit of claim 25, wherein the amine compound is morpholine.

31. The kit of claim 25, wherein the nonwoven support is optionally functionalized with monomers selected from glycidyl methacrylate (GMA) and N-vinyl-2-pyrrolidone (NVP) to enhance the binding capacity and specificity for cell-free polynucleic acids.

32. The kit of claim 25, wherein the nonwoven support is integrated into a device such as a pipet tip, a column, or a filtration unit for streamlined processing of plasma samples.

33. The kit of claim 25, further comprises calibration standards for quantifying the yield and purity of cell- free polynucleic acids.

34. The kit of claim 25, further comprises instructional materials detailing a step-by-step procedure for using the kit, including sample preparation, DNA binding, washing, and elution protocols.

35. The kit of claim 25, wherein the cell-free polynucleic acids are low abundance, low molecular weight cell-free DNA (cfDNA).

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