Liquid biopsy cell enrichment filter structure

Abstract

The present application relates to the technical field of biomedical engineering, in particular to a liquid biopsy cell enrichment filter structure. The structure comprises a plurality of layers of through-hole array filter membranes, which are arranged in order from top to bottom by a large-pore layer, a medium-pore layer and a small-pore layer, and each layer of filter holes corresponds to form a through-communication structure with decreasing pore size. A support layer is arranged between adjacent pore layers to provide mechanical support, and a capture layer covers the support layer for filtering and capturing target cells. The filter membrane edge is integrated and sealed as a whole structure. The structure specifically enriches circulating tumor cells through a multi-stage filtration mechanism, reduces white blood cell interference, solves the problems of white blood cell mis-enrichment, cell release difficulty, filter membrane blockage and difficulty in maintaining cell activity of traditional filter membranes, and improves the recovery rate and purity of CTC.

Description

Technical Field

[0001] This utility model relates to the field of biomedical engineering technology, and more specifically, to a liquid biopsy cell enrichment filter membrane structure. Background Technology

[0002] Circulating tumor cells (CTCs) are rare cells found in very low abundance in peripheral blood. They detach from solid tumors and enter the body's circulatory system. Studies have shown that CTCs exhibit extensive molecular heterogeneity, which persists throughout the entire course of disease development. As a sustainable and readily available source of tumor cells, CTCs are considered a target for "liquid biopsies" of both primary and metastatic tumors.

[0003] Liquid biopsy-based CTC separation and enrichment methods can be mainly divided into two categories: physical property-based separation and bioaffinity-based separation. Physical property-based CTC separation methods can preserve the heterogeneity of target cells and achieve enrichment of all target cells. Among them, microfiltration based on cell size separation is widely recognized as a promising method for achieving high-throughput separation and meeting the needs of processing actual clinical samples.

[0004] However, existing filter membrane structures have several key technical problems: 1. Severe false enrichment of white blood cells: Most filter membranes adopt a single pore size design (usually 10-12μm), which can retain CTC (15-30μm in diameter), but easily retains large white blood cells (8-15μm in diameter), leading to interference in subsequent detection; 2. Difficulty in releasing cells: The presence of adhesion forces results in low efficiency in releasing captured tumor cells from the filter membrane; 3. Membrane clogging problem: Traditional membrane filters are prone to clogging and cell aggregation during the filtration process, which reduces the recovery rate and purity of CTCs; 4. Cell viability is difficult to maintain: Cells are easily damaged during enrichment and release, affecting subsequent analysis.

[0005] Therefore, there is an urgent need for a filter membrane structure that can specifically enrich CTCs, reduce leukocyte interference, and ensure cell integrity. Utility Model Content

[0006] To address the shortcomings of existing methods, the purpose of this invention is to provide a liquid biopsy cell enrichment filter membrane structure that can specifically enrich CTCs, reduce leukocyte interference, and ensure cell integrity, thereby solving the problems of large leukocyte interference, difficulty in cell release, severe clogging, and low cell activity in the existing filter membrane during CTC enrichment.

[0007] To achieve the above objectives, the technical solution adopted by this utility model is as follows: A liquid biopsy cell enrichment filter membrane structure includes a multilayer porous array filter membrane, a support layer, a capture layer, and a filter membrane seal; The multi-layer porous array filter membrane is used to achieve multi-stage filtration of biopsy cells, including a macroporous layer, a mesoporous layer and a microporous layer; the macroporous layer is located in the upper part of the filter membrane, the mesoporous layer is located in the middle of the filter membrane, and the microporous layer is located in the lower part of the filter membrane; the filter pores of each layer correspond one-to-one, forming a through-connected structure with decreasing pore size from top to bottom; The support layer is disposed between two adjacent perforated layers to provide mechanical support for the perforated layers; The capture layer is located between two adjacent porous layers and encloses the support layer, and is used to filter and capture target cells. The filter membrane edge seal covers the periphery of the multi-layer through-hole array filter membrane, and is used to integrate and seal the filter membrane as a whole.

[0008] Furthermore, the surfaces of the macroporous layer, mesoporous layer, and microporous layer are all modified with biomimetic micro / nano structures, including a composite coating of nano zinc oxide and chitosan oligosaccharide.

[0009] Furthermore, the thickness of the macroporous layer, mesoporous layer and microporous layer is 5-10 μm, and the filter membrane is integrally formed from medical-grade parylene through chemical vapor deposition.

[0010] Furthermore, the filter pores of the macroporous layer are circular with a diameter of 20-25 μm; the filter pores of the mesoporous layer are circular with a diameter of 8-12 μm; and the filter pores of the microporous layer are hexagonal with a diameter of 5-8 μm.

[0011] Furthermore, the surface of the macroporous layer is evenly provided with multiple sets of circular microgrooves, and the filter holes within the range of the microgrooves are arranged in a windmill-shaped spiral structure.

[0012] Furthermore, the capture layer is a thermosensitive hydrogel layer composed of poly(N-isopropylacrylamide) and its derivatives.

[0013] Furthermore, the support layer is composed of a uniformly arrayed support pins, with both ends of the pins bonded to the perforated layers on both sides.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention first traps CTCs and large-sized impurities through a macroporous layer (20-25μm), then removes the remaining large-sized white blood cells through a mesoporous layer (8-12μm), and finally discharges small-sized white blood cells through a microporous layer (5-8μm), forming a progressive white blood cell removal path.

[0015] 2. The capture layer thermosensitive hydrogel of this invention not only physically intercepts CTCs that leak through the macroporous layer, but its surface amino groups can also specifically adsorb to the carboxyl groups of CTC cell membranes, further reducing CTC loss and improving CTC purity.

[0016] 3. The evenly distributed circular microgrooves and windmill-shaped spiral arrangement of filter holes on the surface of the macroporous layer can guide the liquid to flow along a preset path, reducing local turbulence and cell accumulation.

[0017] 4. The support layer maintains a stable spacing between adjacent pore layers, avoiding flow channel blockage caused by filter membrane adhesion.

[0018] Of course, any product implementing this utility model does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of this utility model, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the overall structure of this utility model.

[0021] Figure 2 This is a schematic diagram of the disassembled structure of this utility model.

[0022] Figure 3 This is a cross-sectional view of the present invention.

[0023] Figure 4 This is a schematic diagram of the structure of the large-pore layer in this utility model.

[0024] Figure 5 This is a partial structural diagram of the mesoporous layer in this utility model.

[0025] Figure 6 This is a partial structural diagram of the small pore layer in this utility model.

[0026] In the figure: 1. Macropore layer; 11. Microgroove; 2. Filter membrane sealing edge; 3. Capture layer; 4. Support layer; 5. Mesopore layer; 6. Micropore layer. Detailed Implementation

[0027] The technical solutions of this utility model will be clearly and completely described below with reference to the embodiments of this utility model. Obviously, the described embodiments are only some embodiments of this utility model, and not all embodiments. Based on the embodiments of this utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of this utility model.

[0028] Example 1: like Figures 1 to 6 As shown, this embodiment provides a liquid biopsy cell enrichment filter membrane structure, including a multilayer porous array filter membrane, a support layer 4, a capture layer 3, and a filter membrane sealing edge 2.

[0029] The multilayer porous array filter membrane consists of three polymer membrane layers with precise pore size gradients. The filter pores of each filter membrane adopt a one-to-one vertical through structure, forming a fluid channel with continuously decreasing pore size from top to bottom, which is used to achieve multi-stage filtration of biopsy cells. It includes a macroporous layer 1, a mesoporous layer 5, and a microporous layer 6. The macroporous layer 1 is located in the upper part of the filter membrane, the mesoporous layer 5 is located in the middle of the filter membrane, and the microporous layer 6 is located in the lower part of the filter membrane.

[0030] Among them, the filter pores of macroporous layer 1 are circular in shape and have a pore size of 22μm. This pore size is designed to be 1.0 to 1.5 times the maximum diameter of the target tumor cells, which can initially intercept and distribute fluids for large cell clusters and super-large tumor cells, and avoid blockage at the inlet. The surface of the macroporous layer 1 is evenly distributed with multiple sets of circular microgrooves 11, and the filter pores within the range of the microgrooves 11 are arranged in a windmill-shaped spiral structure, which can make the cells disperse evenly, reduce the pressure of the fluid on the cells, reduce the formation of cell clusters, and improve the clearance efficiency of white blood cells.

[0031] In addition, the filter pores of the mesoporous layer 5 are circular in shape with a pore size of 10 μm, which is slightly smaller than the minimum diameter of most circulating tumor cells—12 μm, but larger than the maximum diameter of most white blood cells—8 μm. This allows for precise interception of target circulating tumor cells while allowing the vast majority of white blood cells, red blood cells, and platelets to pass through.

[0032] Furthermore, the filter pores of the micropore layer 6 are hexagonal in shape, achieving maximum opening ratio and structural stability. The pore size is 6μm, which can ensure that all residual small-volume white blood cells, such as lymphocytes (6-10μm in diameter) and red blood cells (6-8μm in diameter), can pass through smoothly, and provide mechanical support for the upper structure to prevent deformation under fluid pressure.

[0033] In this embodiment, the thickness of macroporous layer 1, mesoporous layer 5 and microporous layer 6 are all 8μm to ensure short-range cell migration and rapid passage. The filter membrane is integrally formed from medical-grade parylene through chemical vapor deposition, and has excellent biocompatibility, chemical inertness and mechanical flexibility.

[0034] The support layer 4 is located between two adjacent perforated layers to provide mechanical support for the perforated layers. The support layer 4 is composed of a uniform array of support pins, with its two ends bonded to the perforated layers on both sides.

[0035] The capture layer 3 is located between two adjacent porous layers and encapsulates the support layer 4. It is used to filter and capture target cells. The capture layer 3 is a thermosensitive hydrogel layer composed of poly(N-isopropylacrylamide) and its derivatives. When the temperature is below 32°C, the hydrogel swells and generates sufficient mechanical force to release the captured cells, thereby achieving non-destructive cell recovery.

[0036] When the temperature is above 32°C, the hydrogel is in a hydrophobic collapse state, with the effective pore size at its maximum, which does not affect cell retention. When the temperature is below 32°C, such as 25°C, the hydrogel absorbs water and swells, with a volume expansion rate of more than 150%, which reduces the effective pore size by about 2μm and generates an expansion stress of about 0.5nN / μm² on the adhered cells, gently pushing them away from the pore wall and achieving non-destructive release.

[0037] The filter membrane edge seal 2 is wrapped around the periphery of the multi-layer through-hole array filter membrane to integrate and seal the filter membrane as a whole.

[0038] The surfaces of macroporous layer 1, mesoporous layer 5, and microporous layer 6 are all modified with biomimetic micro / nano structures, including a composite coating of nano-zinc oxide and chitosan oligosaccharides. This design draws on the advantages of leukocyte filtration membranes, providing strong adhesion and high stability. During use, it prevents substances from being washed out or detached from the membrane, effectively preventing blood contamination. Simultaneously, this modification alleviates or prevents blood agglomeration and clears blockages in the membrane pores.

[0039] Example 2: This embodiment provides the working principle of a liquid biopsy cell enrichment filter membrane structure:

[0040] The blood sample to be tested enters the macroporous layer 1 from the upper part of the filter membrane. Guided by the microgrooves 11 and the windmill-shaped spiral arrangement of filter pores, the sample liquid flows along the preset path. Large impurities are intercepted by the 22μm pore size filter pores. At the same time, CTCs, because their size is larger than the filter pores, are initially intercepted on the lower surface of the macroporous layer 1. Some small white blood cells enter the mesoporous layer 5 with the liquid. The liquid entering the mesoporous layer 5 is filtered through a 10μm pore size filter, and white blood cells with a diameter greater than 10μm are retained, further reducing interference in subsequent detection. At this time, the liquid mainly contains CTCs and a small number of small white blood cells, and the small white blood cells enter the microporous layer 6 with the liquid. When the trapping layer 3 below the mesoporous layer 5 comes into contact with the liquid, a small number of CTCs that are not trapped by the macroporous layer 1 are physically trapped by the three-dimensional network structure of the hydrogel. At the same time, the amino groups on the surface of the hydrogel and the carboxyl groups on the surface of the CTC cell membrane are specifically adsorbed, which improves the trapping efficiency. Small white blood cells that enter the microporous layer 6 are discharged from the filter membrane through 6μm regular hexagonal filter pores, achieving efficient separation of CTCs and white blood cells. After enrichment, the filter membrane is placed in a constant temperature environment of 20°C. The thermosensitive hydrogel of the capture layer 3 quickly swells into a liquid state, loses its physical retention and adsorption capacity, and CTCs detach from the filter membrane under gravity and slight rinsing.

[0041] Obviously, the above embodiments of this utility model are merely examples for clearly illustrating this utility model, and are not intended to limit the implementation of this utility model. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. Any obvious variations or modifications derived from the technical solutions of this utility model are still within the protection scope of this utility model.

Claims

1. A liquid biopsy cell enrichment filter membrane structure, characterized in that, include: A multi-layer perforated array filter membrane is used to achieve multi-stage filtration of biopsy cells, including a macroporous layer (1), a mesoporous layer (5) and a microporous layer (6); the macroporous layer (1) is located in the upper part of the filter membrane, the mesoporous layer (5) is located in the middle part of the filter membrane, and the microporous layer (6) is located in the lower part of the filter membrane; the filter pores of each layer correspond one-to-one, forming a through-connected structure with decreasing pore size from top to bottom; A support layer (4) is provided between two adjacent perforated layers to provide mechanical support for the perforated layers; The capture layer (3) is located between two adjacent pore layers and encloses the support layer (4) inside, and is used to filter and capture target cells; The filter membrane edge sealing (2) is wrapped around the periphery of the multi-layer through-hole array filter membrane and is used to integrate and seal the filter membrane as a whole.

2. The liquid biopsy cell enrichment filter membrane structure according to claim 1, characterized in that: The surfaces of the macroporous layer (1), mesoporous layer (5) and microporous layer (6) are all modified with biomimetic micro-nano structures, including a composite coating of nano zinc oxide and chitosan oligosaccharide.

3. The liquid biopsy cell enrichment filter membrane structure according to claim 1, characterized in that: The thickness of the macroporous layer (1), mesoporous layer (5) and microporous layer (6) is 5-10 μm. The filter membrane is integrally formed from medical-grade parylene through chemical vapor deposition.

4. The liquid biopsy cell enrichment filter membrane structure according to claim 1, characterized in that: The macroporous layer (1) has circular pores with a diameter of 20-25 μm; the mesoporous layer (5) has circular pores with a diameter of 8-12 μm; and the microporous layer (6) has regular hexagonal pores with a diameter of 5-8 μm.

5. The liquid biopsy cell enrichment filter membrane structure according to claim 1, characterized in that: The surface of the macroporous layer (1) is evenly provided with multiple sets of circular microgrooves (11), and the filter holes within the range of the microgrooves (11) are arranged in a windmill-shaped spiral structure.

6. The liquid biopsy cell enrichment filter membrane structure according to claim 1, characterized in that: The capture layer (3) is a thermosensitive hydrogel layer composed of poly(N-isopropylacrylamide) and its derivatives.

7. The liquid biopsy cell enrichment filter membrane structure according to claim 1, characterized in that: The support layer (4) is composed of a uniform array of support pins, with its two ends bonded to the porous layers on both sides.

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