A device and method for extracting cell-free DNA based on electrophoresis-dialysis coupling

Abstract

This invention relates to the field of biological sample pretreatment and molecular diagnostics, and discloses a cell-free DNA extraction device and method based on electrophoresis-dialysis coupling. The device includes an outer container, a middle container, an inner container, and a collection container. The middle container is disposed within the outer container, and a gap exists between the middle and outer containers to form a first electrode cavity, within which an anode is located. The inner container is disposed within the middle container, and a gap exists between the inner and middle containers to form a second electrode cavity, within which a cathode is located. The collection container has open ends, with a connection end and a filter end from top to bottom. The connection end is detachably connected to the middle container and communicates with the inner container. The filter end communicates with the first electrode cavity and is equipped with a dialysis membrane. This invention enables cell-free DNA to achieve directional migration, desalting, and enrichment under an electric field, and is suitable for rapid cfDNA extraction from complex biological samples.

Description

Technical Field

[0001] This invention relates to the field of biological sample pretreatment and molecular diagnostics, and in particular to a device and method for extracting cell-free DNA based on electrophoresis-dialysis coupling. Background Technology

[0002] Currently, extracting cell-free DNA (cfDNA) from biological samples (such as plasma and urine) is a crucial prerequisite for cutting-edge medical diagnostics such as liquid biopsy and non-invasive prenatal testing. However, existing mainstream cfDNA extraction technologies, such as centrifugation column methods and magnetic bead methods, have significant shortcomings. First, these methods are cumbersome, involving multiple pipetting, centrifugation, or magnetic separation steps, and are time-consuming (typically 30 minutes to 2 hours), making it difficult to achieve high-throughput and automated processing. Second, these methods rely on solid-phase materials such as silica membranes or magnetic beads to adsorb, wash, and elute DNA. This process not only easily leads to the loss of low-concentration cfDNA but also introduces mechanical shear forces that damage large cfDNA fragments, reducing recovery rates and product integrity. In addition, commercially available kits are expensive, especially when processing large volumes of samples (such as 5-10 mL of urine), placing a heavy economic burden on the manufacturer. More importantly, traditional methods struggle to effectively remove small-molecule salts and impurities from the sample during extraction, and high-salt environments can interfere with downstream molecular detection methods such as PCR. Therefore, there is an urgent need for a new cfDNA extraction technology that is simple to operate, fast, low-cost, has a high recovery rate, and can simultaneously achieve desalting. Summary of the Invention

[0003] The purpose of this invention is to provide a cell-free DNA extraction device and method based on electrophoresis-dialysis coupling, aiming to solve or improve at least one of the above-mentioned technical problems.

[0004] To achieve the above objectives, the present invention provides the following solution: The present invention provides a cell-free DNA extraction device based on electrophoresis-dialysis coupling, comprising an outer container, a middle container, an inner container, and a collection container; The intermediate container is disposed in the outer container, and there is a gap between the intermediate container and the outer container to form a first electrode cavity, and an anode is disposed in the first electrode cavity; The inner container is disposed in the middle container, and there is a gap between the inner container and the middle container to form a second electrode cavity, and a cathode is disposed in the second electrode cavity; The collection container is open at both ends and, from top to bottom, is a connection end and a filter end. The connection end is detachably connected to the middle container and communicates with the inner container. The filter end is connected to the first electrode cavity and is provided with a dialysis membrane.

[0005] Optionally, the outer container, the middle container, the inner container, and the collection container are arranged coaxially.

[0006] Optionally, the connecting end of the collection container is detachably connected to the intermediate container via a threaded structure.

[0007] Optionally, the inner container is a porous isolation structure, comprising a porous polyethylene tube and an agarose layer filling its pores.

[0008] Optionally, both the anode and the cathode are liquid electrodes.

[0009] Optionally, the molecular weight cutoff of the dialysis membrane is in the range of 0.5–100 kDa.

[0010] Optionally, the intermediate container and the inner container are connected by adhesive.

[0011] Optionally, the bottom surface of the outer container is provided with multiple slots, and the bottom surface of the middle container is fixedly connected with multiple insert rods, with each insert rod corresponding to one of the multiple slots and fitting with a gap.

[0012] This invention also provides a method for extracting cell-free DNA based on electrophoresis-dialysis coupling, comprising the following steps: The biological sample containing free DNA is placed in the inner container; A DC electric field is applied to the anode and the cathode, causing negatively charged free DNA to migrate directionally toward the dialysis membrane under the action of the electric field. Small molecules are filtered through the dialysis membrane, while large molecules are retained and enriched in the collection container, thereby achieving the enrichment and desalting of free DNA.

[0013] Optionally, the voltage of the DC electric field is from 50V to 300V.

[0014] The present invention discloses the following technical effects: This invention utilizes a DC electric field to drive negatively charged cfDNA to migrate directionally to a collection container, without involving any rough operations such as adsorption or washing. This greatly reduces the physical loss and fragmentation risk of cfDNA, ensuring higher recovery rates and integrity of the extracted products. The advantages are particularly evident for extracting low-concentration, large-fragment cfDNA samples.

[0015] In this invention, during electrophoretic migration, cfDNA passes through a dialysis membrane into the collection container. This dialysis membrane effectively blocks cfDNA molecules larger than its molecular weight cutoff, while allowing small salt ions, proteins, and other impurities to pass through and diffuse into the first electrode chamber. This process achieves simultaneous enrichment and desalting purification of cfDNA, and the obtained product can be directly used for downstream enzymatic reactions such as PCR, simplifying the operational procedure.

[0016] This invention, through optimized multi-layer coaxial structure and electric field design, can drive cfDNA to migrate and enrich efficiently from large-volume samples in a short time, significantly faster than traditional multi-step centrifugation or magnetic separation methods.

[0017] This invention features a simple structure, eliminating the need for complex centrifugation equipment or automated workstations. Users simply add samples and buffer solution and apply voltage. Furthermore, the core components of the device are significantly cheaper than commercial extraction kits, making it particularly suitable for clinical testing and research laboratories that require processing large volumes of samples.

[0018] This invention is not only applicable to plasma, but also successfully applied to the extraction of large-volume, low-concentration cfDNA samples such as urine. It has good tolerance to high-salt or complex biological matrices, demonstrating strong sample versatility. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the overall structure of the present invention; Figure 2 This is a cross-sectional view of the present invention; Figure 3 This is a schematic diagram of the inner container structure of the present invention.

[0020] In the diagram: 1. Outer container; 2. Middle container; 3. Inner container; 31. Porous polyethylene pipe; 32. Agarose layer; 4. Collection container; 5. Anode; 6. Cathode; 7. Dialysis membrane; 8. Threaded structure; 9. Slot; 10. Insert rod. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Reference Figures 1 to 3 The present invention provides a cell-free DNA extraction device based on electrophoresis-dialysis coupling, comprising an outer container 1, a middle container 2, an inner container 3, and a collection container 4; The intermediate container 2 is disposed in the outer container 1, and there is a gap between the intermediate container 2 and the outer container 1 to form a first electrode cavity, and an anode 5 is disposed in the first electrode cavity; The inner container 3 is disposed in the middle container 2, and there is a gap between the inner container 3 and the middle container 2 to form a second electrode cavity, and a cathode 6 is disposed in the second electrode cavity; The collection container 4 is open at both ends and from top to bottom are a connection end and a filter end. The connection end is detachably connected to the middle container 2 and communicates with the inner container 3. The filter end is connected to the first electrode cavity and is provided with a dialysis membrane 7.

[0024] In this embodiment, the outer container 1, the middle container 2, the inner container 3, and the collection container 4 are coaxially arranged. This coaxial structure creates a uniform and symmetrical concentric cylindrical electric field within the device. This uniform electric field distribution ensures that the electric force on cfDNA molecules at all radial positions within the sample chamber (inner container 3) is consistent (all axially downwards), thereby enabling parallel, efficient, and concentrated migration of cfDNA to the bottom collection container. This avoids local migration speed differences or migration dead zones caused by uneven electric fields, significantly improving the collection efficiency and consistency of cfDNA enrichment concentration.

[0025] In this embodiment, the connecting end of the collection container 4 is detachably connected to the intermediate container 2 via a threaded structure 8. This detachable connection via the threaded structure provides significant operational convenience and reliability, allowing the experimenter to easily unscrew the collection container 4 after extraction.

[0026] In this embodiment, the inner container 3 is a porous isolation structure, including a porous polyethylene tube 31 and an agarose layer 32 filled in its pores. This structure creates a highly efficient ion-conducting and material-isolating interface. The porous polyethylene tube 31 provides physical support, while the filled agarose layer 32 forms a three-dimensional gel network. This effectively prevents large particulate impurities, cell debris, or microorganisms in the sample chamber from entering the electrode chamber, and conversely, it also prevents bubbles or byproducts generated by the electrode reaction from entering the sample chamber. By limiting the rapid convection and diffusion between the sample and the electrode buffer, a relatively stable low ionic strength and pH environment is maintained in the sample area, which is crucial for the efficient migration of cfDNA under an electric field, avoiding damage to DNA caused by drastic pH changes due to the electrode reaction. The agarose layer 32 can suppress electroosmotic flow and liquid thermal convection, ensuring that cfDNA is mainly driven by electrophoretic force to achieve directional migration.

[0027] In this embodiment, both anode 5 and cathode 6 are liquid electrodes. Using liquid electrodes (i.e., directly using buffer solution as the electrode) instead of traditional solid metal electrodes offers significant advantages. First, it completely avoids the problem of metal ions (such as platinum, gold, copper, etc.) potentially precipitating and contaminating the sample during electrolysis, ensuring the extracted cfDNA has extremely high chemical purity and does not affect downstream sensitive detection. Second, liquid electrodes have a high specific heat capacity, which can more effectively absorb and dissipate the Joule heat generated during electrophoresis, preventing localized overheating that could lead to cfDNA denaturation or the formation of bubbles within the device. Finally, liquid electrodes have a simple configuration, extremely low cost, and are easy to maintain and scale up for application.

[0028] In this embodiment, the molecular weight cutoff of the dialysis membrane is 0.5–100 kDa. The lower limit of 0.5 kDa ensures that small molecule salt ions, nucleotides, peptides, and other impurities can freely pass through the dialysis membrane and be removed, achieving effective desalting. The upper limit of 100 kDa ensures efficient retention of cfDNA, because most cfDNA fragments are between 100-300 bp in length, corresponding to molecular weights much larger than 100 kDa. By selecting a specific cutoff value within this range, the separation and enrichment of cfDNA fragments of different sizes can also be achieved. For example, selecting a lower cutoff value (such as 30 kDa) enriches mononuclear-related DNA, while selecting a higher cutoff value (such as 100 kDa) allows larger DNA-protein complexes to pass through, providing additional selectivity.

[0029] In this embodiment, the intermediate container 2 and the inner container 3 are connected by adhesive bonding. Using adhesive bonding to connect the intermediate container 2 and the inner container 3 significantly improves structural stability and operational reliability. First, the adhesive connection forms a seamless and robust fixed structure, ensuring that the inner container 3 and the intermediate container 2 maintain a precise coaxial alignment during long-term use or repeated disassembly and assembly, thereby maintaining the uniformity and stability of the electric field distribution and avoiding electric field distortion or migration path deviation caused by relative displacement. Second, the adhesive interface provides auxiliary sealing, effectively preventing capillary leakage of sample liquid or buffer solution from the gap between the two containers, reducing the risk of cross-contamination.

[0030] In this embodiment, the inner bottom surface of the outer container 1 has multiple slots 9, and the outer bottom surface of the middle container 2 is fixedly connected to multiple insert rods 10. The multiple insert rods 10 correspond one-to-one with the multiple slots 9 and are fitted with clearance. The corresponding fit between the multiple insert rods 10 and the slots 9 restricts the radial rotation and horizontal offset of the middle container 2 within the outer container 1, ensuring that the two always maintain a precise coaxial relationship, and realizing fast, accurate, and repeatable installation positioning between the outer container 1 and the middle container 2.

[0031] This invention also provides a method for extracting cell-free DNA based on electrophoresis-dialysis coupling, comprising the following steps: Place the biological sample containing free DNA in the inner container 3; A DC electric field is applied to the anode 5 and the cathode 6, causing the negatively charged free DNA to migrate directionally toward the dialysis membrane 7 under the action of the electric field. The small molecules are filtered through the dialysis membrane 7, while the large molecules are retained and enriched in the collection container 4, thereby achieving the enrichment and desalting of free DNA.

[0032] In this embodiment, the DC electric field voltage is 50V to 300V. This voltage range represents the optimal balance between migration efficiency and sample integrity. Below 50V, the driving force of the electric field is too weak, resulting in slow cfDNA migration and prolonged total extraction time, and it is ineffective for large-volume samples or long DNA fragments. Above 300V, the excessively strong electric field generates severe Joule heating, which may cause the liquid inside the device to heat up, leading to thermal degradation of cfDNA, convection interference with migration direction, and even damage to the gel medium or dialysis membrane. The 50-300V voltage range ensures that cfDNA migrates at an ideal speed under a safe, efficient, and stable electric field strength, achieving rapid and complete enrichment.

[0033] Example 1: Extraction of cfDNA from plasma Sample preparation: Collect 5 mL of peripheral blood from healthy volunteers using EDTA anticoagulant tubes. Immediately after blood collection, perform plasma separation. Centrifuge the whole blood at 3000×g for 10 minutes and collect the supernatant plasma. Centrifuge the plasma again at 13,000×g for 5 minutes to remove residual cell debris. Take 5 mL of the supernatant plasma for later use.

[0034] Buffer preparation: Sample buffer: 10 mM Tris-HCl, pH 8.0; Electrode buffer: 50 mM Tris-glycine, pH 8.5.

[0035] Assemble and run the device according to the following steps: Add 5 mL of plasma sample to the inner container 3; add appropriate amounts of electrode buffer to the middle container 2 and the outer container 1; ensure that the agarose-filled porous isolation structure is correctly installed to isolate the sample area from the electrode buffer area; connect the DC power supply and apply a 100V DC voltage; after running for 5 minutes, turn off the power and detect significantly enriched cfDNA in the dialysis cup collection chamber.

[0036] Results: Samples were taken from the dialysis cup collection chamber, and DNA concentration was measured using a Qubit quantitative PCR instrument and a Qubit dsDNA HSAssay Kit. The results showed significantly enriched cfDNA in the dialysis cup collection chamber.

[0037] Example 2: Extraction of cfDNA from urine This embodiment demonstrates the application of the device of the present invention in extracting cfDNA from urine samples. Urine cfDNA detection has significant application value in fields such as the diagnosis of urinary system tumors and the monitoring of kidney disease.

[0038] Sample preparation: Collect 10 mL of morning urine from healthy volunteers, centrifuge the urine at 3,000 × g for 5 minutes to remove cells and impurities; take 5 mL of supernatant for later use.

[0039] Buffer preparation: Sample buffer: 10 mM Tris-HCl, pH 8.0; Electrode buffer: 50 mM Tris-glycine, pH 8.5.

[0040] The device is assembled and operated according to the same steps as in Example 1.

[0041] Results: Samples were taken from the dialysis cup collection chamber, and DNA concentration was determined using a Qubit real-time fluorescence analyzer. The results showed significantly enriched cfDNA in the dialysis cup collection chamber.

[0042] Example 3: Verification of Anion Tracing Molecular Migration In this embodiment, negatively charged amaranth is used as a tracer molecule to observe its directional migration behavior toward the anodic dialysis zone under the action of an electric field in a low ionic strength buffer system, in order to verify the electrophoresis-dialysis coupling effect of the device.

[0043] Experimental principle: Amaranth red is a negatively charged organic dye molecule (molecular weight approximately 604 Da) that migrates towards the anode under the influence of an electric field. By observing the migration behavior of amaranth red, the electric field distribution and dialysis effect of the device of this invention can be verified.

[0044] Experimental method: Prepare a 1% amaranth red solution as a tracer; add the tracer to the inner sample container; use a low ionic strength buffer (1 mM Tris-HCl, pH 8.0); apply a 100 V DC voltage; record the migration distance of the tracer molecule at the front end every minute.

[0045] Experimental results showed that, under the influence of an electric field, amaranth molecules migrated towards the anode at a speed of approximately 2 mm / min. At 5 minutes, the leading edge of the tracer molecules reached the edge of the agarose isolation region and began to enter the dialysis cup collection chamber. This result confirms that the electrophoresis-dialysis coupling mechanism of the device of this invention can effectively drive charged molecules to migrate towards the collection region.

[0046] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cell-free DNA extraction device based on electrophoresis-dialysis coupling, characterized in that, It includes an outer container (1), a middle container (2), an inner container (3), and a collection container (4); The intermediate container (2) is disposed in the outer container (1), and there is a gap between the intermediate container (2) and the outer container (1) to form a first electrode cavity, and an anode (5) is disposed in the first electrode cavity. The inner container (3) is disposed in the middle container (2), and there is a gap between the inner container (3) and the middle container (2) to form a second electrode cavity, and a cathode (6) is disposed in the second electrode cavity. The collection container (4) is open at both ends and from top to bottom are a connection end and a filter end, respectively. The connection end is detachably connected to the middle container (2) and communicates with the inner container (3). The filter end is connected to the first electrode cavity and is provided with a dialysis membrane (7).

2. The cell-free DNA extraction device based on electrophoresis-dialysis coupling according to claim 1, characterized in that, The outer container (1), the middle container (2), the inner container (3), and the collection container (4) are arranged coaxially.

3. The cell-free DNA extraction device based on electrophoresis-dialysis coupling according to claim 1, characterized in that, The connecting end of the collection container (4) is detachably connected to the intermediate container (2) via a threaded structure (8).

4. The cell-free DNA extraction device based on electrophoresis-dialysis coupling according to claim 1, characterized in that, The inner container (3) is a porous isolation structure, including a porous polyethylene tube (31) and an agarose layer (32) filled in its pores.

5. The cell-free DNA extraction device based on electrophoresis-dialysis coupling according to claim 1, characterized in that, Both the anode (5) and the cathode (6) are liquid electrodes.

6. The cell-free DNA extraction device based on electrophoresis-dialysis coupling according to claim 1, characterized in that, The molecular weight cutoff of the dialysis membrane is 0.5–100 kDa.

7. The method for extracting cell-free DNA based on electrophoresis-dialysis coupling according to claim 1, characterized in that, The intermediate container (2) and the inner container (3) are connected by adhesive.

8. The method for extracting cell-free DNA based on electrophoresis-dialysis coupling according to claim 1, characterized in that, The outer container (1) has multiple slots (9) on its inner bottom surface, and the middle container (2) has multiple plug rods (10) fixedly connected to its outer bottom surface. The multiple plug rods (10) correspond one-to-one with the multiple slots (9) and are fitted with a gap.

9. A method for extracting cell-free DNA based on electrophoresis-dialysis coupling, wherein the cell-free DNA extraction device based on electrophoresis-dialysis coupling according to any one of claims 1-8 is characterized in that, Includes the following steps: The biological sample containing free DNA is placed in the inner container (3); A DC electric field is applied to the anode (5) and the cathode (6) to cause the negatively charged free DNA to migrate toward the dialysis membrane (7) under the action of the electric field. The small molecules are filtered through the dialysis membrane (7) so that the large molecules are retained and enriched in the collection container (4), thereby achieving the enrichment and desalting of free DNA.

10. The method for extracting cell-free DNA based on electrophoresis-dialysis coupling according to claim 9, characterized in that, The voltage of the DC electric field is 50V to 300V.

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