Microfluidic circulating tumor cell capturing method based on height gradient
By adopting highly gradient coupled microarray structure and modified polydimethylsiloxane layer on the microfluidic chip, the inefficient capture and recovery of circulating tumor cells in the blood is solved, and efficient cell isolation and recovery is achieved , improves the accuracy and efficiency of cancer fluid biopsy.
Patent Information
- Application Number
- CN202411520366.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2025-01-14
AI Technical Summary
The prior art is difficult to efficiently isolate and recover circulating tumor cells, especially in the context of blood, resulting in low capture and recovery rates, affecting the accuracy and efficiency of cancer fluid biopsy.
A highly gradient-based microfluidic chip is designed using a polysiloxane layer and a modified polydimethylsiloxane layer, combining highly gradient-coupled microarray structures and hydrophilic groups for capture and recycling cycles Tumor cells improve capture and recovery efficiency through optimized preparation processes.
A capture rate of more than 73% and a recovery rate of 82% were achieved, which significantly improved the separation efficiency of circulating tumor cells, reduced the aggregation and adhesion of biological particles, and enhanced the dispersion effect of microfluidic chips on cells.
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Figure CN119307352A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biophysical technology, and in particular to a microfluidic circulating tumor cell capturing method based on height gradient. Background Art
[0002] Circulating tumor cells originate from solid tumors and then enter the blood directly by crossing the endothelium, or indirectly through the lymphatic system. Researchers have long speculated that circulating tumor cells are the main way for cancer to metastasize away from its origin. Therefore, they are a key factor in the disease process, and their presence in peripheral blood makes cancer liquid biopsy more convenient. Although circulating tumor DNA and exosomes also provide sources for the detection and genotyping of tumor-specific nucleic acids in the blood, circulating tumor cells provide unique possibilities. Therefore, researchers have been working to improve methods for isolating circulating tumor cells from the blood to fully tap their unique and rich biological information in order to maximize the benefits in anti-cancer treatment.
[0003] In order to achieve liquid biopsy based on circulating tumor cells, isolating a sufficient number of high-purity circulating tumor cells is a prerequisite. Currently, there are a variety of methods available for the separation of circulating tumor cells, which can be divided into three categories: biological enrichment, physical enrichment, and unbiased enrichment of all nucleated cells. Since circulating tumor cells are very rare, usually there is only 1 circulating tumor cell among billions of blood cells, the development of a suitable, high-throughput and high-precision circulating tumor cell separation method remains an urgent issue to be solved. Summary of the invention
[0004] The purpose of the present invention is to provide a microfluidic circulating tumor cell capture method based on height gradient to improve the capture rate and recovery rate of circulating tumor cells by microfluidic chip and realize label-free and high-purity capture of circulating tumor cells.
[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A microfluidic chip based on height gradient comprises a polysiloxane layer, a glass layer, a fluid region formed between the polysiloxane layer and the glass layer, and a chip inlet and a chip outlet connected to the fluid region; a height gradient coupling microarray structure is provided on the surface of the polysiloxane layer facing the fluid region; the longitudinal configuration of the height gradient coupling microarray structure is stepped, comprising at least 3 height gradients, and the smallest height gradient size is 9-11 μm.
[0006] The present invention designs a microfluidic chip with a height gradient coupled microarray structure according to the size and deformability of cells, so as to realize efficient capture and recovery of circulating tumor cells in different regions; the microfluidic chip sets different height gradients for the two states of cell clusters and single cells, captures cell clusters at a height gradient greater than 20 μm, and captures single cells at a height gradient greater than 10 μm; and the microfluidic chip is combined with a microarray structure design on the height gradient, and its geometric shape tends to capture only single cells, and can realize observation at the same position while capturing, preventing the position migration of single cells.
[0007] Preferably, the lateral configuration of the height gradient coupling microarray structure is wavy.
[0008] Preferably, the diameter of the chip inlet is 0.5-1.5 mm, and the diameter of the chip outlet is 0.5-1.5 mm.
[0009] Preferably, the polysiloxane layer includes a polydimethylsiloxane layer and / or a modified polydimethylsiloxane layer, and the modified polydimethylsiloxane layer has a naphthyl group and a carbonyl group.
[0010] More preferably, in the preparation of the polydimethylsiloxane layer, a highly gradient coupled microarray structure mold is first made on a silicon wafer, and then polydimethylsiloxane and a curing agent are mixed and poured on the mold for reaction to obtain the polydimethylsiloxane layer.
[0011] More preferably, the preparation of the polydimethylsiloxane layer is specifically as follows: First, SU-8 photoresist is evenly coated on a 2-8 inch silicon wafer to form a SU-8 photoresist layer on the surface of the silicon wafer, and the SU-8 photoresist layer is spin-coated at a speed of 2000-4000 rpm for 0.5-3 min, baked at 60-100°C for 0.5-2 h, and then the silicon wafer is exposed to ultraviolet light for 2-5 min to cure the glue layer. This step is repeated to form a total thickness of 30-90 μm on the surface of the silicon wafer. SU-8 photoresist structure, using ultraviolet lithography technology to make a highly gradient coupled microarray structure mold on a silicon wafer, mixing polydimethylsiloxane and a curing agent, stirring at 2000-4000rpm for 20-40min, standing in a dark place for 20-40min, continuing to stir evenly until the mixture is filled with fine and uniform bubbles, pouring the mixture on the mold, placing it in a vacuum box for 30-60min of degassing treatment, reacting under ultraviolet light for 80-120s, and then placing it in an oven at 60-100℃ and heating for 60-120min. After complete curing, a polydimethylsiloxane layer is obtained.
[0012] More preferably, the mass ratio of polydimethylsiloxane to curing agent is 1:0.1-1.
[0013] More preferably, in the preparation of the modified polydimethylsiloxane layer, a highly gradient coupled microarray structure mold is first made on a silicon wafer, and then polydimethylsiloxane, isooctyl acrylate, a vinyl monomer substance, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide and a curing agent are mixed and poured on the mold for reaction to obtain a modified polydimethylsiloxane layer; the vinyl monomer substance includes at least tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone.
[0014] More preferably, the preparation of the modified polydimethylsiloxane layer is specifically as follows: First, SU-8 photoresist is evenly coated on a 2-8 inch silicon wafer to form a SU-8 photoresist layer on the surface of the silicon wafer, and the SU-8 photoresist layer is spin-coated at a speed of 2000-4000 rpm for 0.5-3 min, baked at 60-100°C for 0.5-2 h, and then the silicon wafer is exposed to ultraviolet light for 2-5 min to cure the glue layer. This step is repeated to form a total thickness of 30-90 μm on the surface of the silicon wafer. SU-8 photoresist structure, using ultraviolet lithography technology to make a highly gradient coupled microarray structure mold on a silicon wafer, polydimethylsiloxane, isooctyl acrylate, tetrahydrofurfuryl acrylate, 2-naphthyl vinyl ketone, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide and a curing agent are mixed, stirred at 2000-4000 rpm for 20-40 minutes, and allowed to stand in a dark place for 20-40 minutes. The mixture is stirred evenly until it is filled with fine and uniform bubbles. The mixture is poured on the mold, placed in a vacuum box for 30-60 minutes of degassing treatment, reacted under ultraviolet light for 80-120 seconds, and then placed in an oven at 60-100°C for heating for 60-120 minutes. After complete curing, a modified polydimethylsiloxane layer is obtained. The invention uses a light-initiated free radical polymerization reaction to obtain a modified polydimethylsiloxane having an interpenetrating polymer network structure. The modified polydimethylsiloxane is used to prepare a material for a microfluidic chip by introducing hydrophilic groups, such as vinyl and carbonyl, into the polydimethylsiloxane body, thereby helping to enhance the dispersion effect of the microfluidic chip on biological particles, thereby improving the capture rate and recovery rate of circulating tumor cells by the microfluidic chip.
[0015] More preferably, the volume ratio of polydimethylsiloxane to isooctyl acrylate is 1:1-2.
[0016] More preferably, the usage ratio of polydimethylsiloxane and tetrahydrofurfuryl acrylate is 1 mL: 0.1-1 g.
[0017] More preferably, the usage ratio of polydimethylsiloxane and 2-naphthylene ketone is 1 mL: 0.1-1 g.
[0018] More preferably, the usage ratio of polydimethylsiloxane and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide is 1 mL: 0.1-0.3 g.
[0019] More preferably, the mass ratio of polydimethylsiloxane to curing agent is 1:0.1-1.
[0020] More preferably, in the preparation of the modified polydimethylsiloxane layer, ethyl vinyl ether is further added as a vinyl monomer substance to prepare the modified polydimethylsiloxane layer, and the amount ratio of polydimethylsiloxane to ethyl vinyl ether is 1 mL: 0.1-0.5 g. Introducing ethyl vinyl ether into the polydimethylsiloxane body to obtain a modified polydimethylsiloxane containing a large number of polar groups can further improve the capture rate and recovery rate of circulating tumor cells in the microfluidic chip.
[0021] Preferably, in the preparation of the microfluidic chip, the surfaces of the polysiloxane layer and the glass layer facing the fluid region are first treated with air plasma, and then the polysiloxane layer and the glass layer are bonded to obtain the microfluidic chip.
[0022] Preferably, the method for preparing the microfluidic chip comprises: First, the surfaces of the polysiloxane layer and the glass layer facing the fluid area are treated with air plasma, and then the polysiloxane layer and the glass layer are bonded, and then a chip inlet with a diameter of 0.5-1.5mm and a chip outlet with a diameter of 0.5-1.5mm are configured at both ends to obtain a microfluidic chip.
[0023] The present invention also provides a method for capturing circulating tumor cells in a microfluidic system based on a height gradient, comprising injecting a fluid into a microfluidic chip to capture circulating tumor cells, and recovering the circulating tumor cells by backwashing, wherein the fluid contains circulating tumor cells.
[0024] Preferably, the amount of fluid used is 0.5-2 mL.
[0025] Preferably, the flow rate of the fluid is 0.2-2 mL / h.
[0026] Preferably, a method for capturing circulating tumor cells by microfluidics based on height gradient is as follows: Before the experiment, the flow channel of the microfluidic chip was cleaned with ethanol for 2-4 times, and then rinsed with deionized water or phosphate buffer for 2-4 times. 0.2-2mL of fluid was injected into the microfluidic chip through the left inlet at a flow rate of 0.2-1mL / h using a syringe pump and a syringe. Biological particles larger than 10μm were captured on the height gradient of the microfluidic chip and could be directly observed using a fluorescent inverted microscope. The remaining biological particles smaller than 10μm were recovered from the outlet, and the inlet and outlet were swapped, so that circulating tumor cells could be recovered by reverse flushing.
[0027] The present invention uses a vinyl monomer substance containing a hydrophilic group to modify polydimethylsiloxane to obtain a modified polydimethylsiloxane layer, and then uses the modified polydimethylsiloxane layer to prepare a microfluidic chip, and the prepared microfluidic chip is used to capture circulating tumor cells, so it has the following beneficial effects: the modified polydimethylsiloxane layer after modification has a large number of hydrophilic groups, which helps to enhance the dispersion effect of biological particles and reduce the aggregation and adhesion of biological particles when capturing circulating tumor cells in the microfluidic chip, so that the capture rate of circulating tumor cells in the microfluidic chip reaches more than 73%, and the recovery rate reaches more than 82%. Therefore, the present invention is a microfluidic circulating tumor cell capture method based on height gradient with high capture rate and high recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a schematic diagram of the structure of the microfluidic chip prepared in Example 1.
[0029] Figure 2 This is the distribution diagram of the height gradient area in the microfluidic chip prepared in Example 1.
[0030] Figure 3 Schematic diagram of the capture principle of the microfluidic chip prepared in Example 1.
[0031] Figure 4 The image of circulating tumor cells captured by the microfluidic chip prepared in Example 1.
[0032] Figure 5 This is the infrared spectrum of the modified polydimethylsiloxane layer prepared in Example 5. DETAILED DESCRIPTION
[0033] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
[0035] Embodiment 1: S1. Preparation of a polydimethylsiloxane layer, comprising: First, SU-8 photoresist was uniformly coated on a 4-inch silicon wafer to form a SU-8 photoresist layer with a thickness of 10 μm, and then the layer was spin-coated at a rotation speed of 3000 rpm for 1 min, baked at 80°C for 1 hour, and then the silicon wafer was exposed to ultraviolet light for 3 min to cure the layer. This step was repeated twice to form a three-layer SU-8 photoresist structure with a total thickness of 30 μm. The thickness of each SU-8 photoresist layer was 10 μm. A highly gradient coupled microarray structure mold was made on the silicon wafer using ultraviolet lithography technology. The longitudinal configuration of the highly gradient coupled microarray structure was stepped, including three height zones. The size of height region I is 30 μm, the size of height region II is 20 μm, the size of height region III is 10 μm, and the longitudinal configuration of the gradient is sinusoidal. Then, the polydimethylsiloxane is mixed with the curing agent and stirred evenly until the mixture is filled with fine and uniform bubbles. The mixture is poured on the mold and placed in a vacuum box for degassing for 40 minutes. Then, it is placed in an oven at 85°C and heated for 90 minutes. After the polydimethylsiloxane is completely cured, the polydimethylsiloxane layer is peeled off from the mold with a blade to obtain a polydimethylsiloxane layer with a size of 30 mm×15 mm×5 mm.
[0036] In this embodiment, the polydimethylsiloxane and the curing agent are components in SYLGARD™ 184 Silicone Elastomer Kit produced by Dow Chemical Company, and the mass ratio of the polydimethylsiloxane to the curing agent is 1:0.1.
[0037] S2. Preparation of microfluidic chip, including: The glass was cut into a size of 50 mm × 30 mm × 1 mm. The polydimethylsiloxane layer obtained in step S1 and the surface of the glass layer facing the fluid area were first treated with air plasma. The polydimethylsiloxane layer obtained in step S1 and the glass layer were then bonded together. A chip inlet and a chip outlet with a diameter of 1 mm were then arranged at both ends to obtain a microfluidic chip.
[0038] S3. Capture of circulating tumor cells, including: Before the experiment, the flow channel of the microfluidic chip was cleaned with ethanol for 3 times and then rinsed with deionized water or phosphate buffer for 3 times. The fluid containing circulating tumor cells was injected into the microfluidic chip through the left inlet at a rate of 1 mL / h using an injection pump and a syringe. Biological particles larger than 10 μm were captured on the microstructure gradient and could be directly observed using a fluorescent inverted microscope. The remaining biological particles smaller than 10 μm were recovered from the outlet. The inlet and outlet were then swapped, and the circulating tumor cells could be recovered by reverse flushing.
[0039] Embodiment 2: S1. Preparation of a modified polydimethylsiloxane layer, comprising: First, SU-8 photoresist was uniformly coated on a 4-inch silicon wafer to form a SU-8 photoresist layer with a thickness of 10 μm, and then the layer was spin-coated at a rotation speed of 3000 rpm for 1 min, baked at 80°C for 1 hour, and then the silicon wafer was exposed to ultraviolet light for 3 min to cure the adhesive layer. This step was repeated twice to form a three-layer SU-8 photoresist structure with a total thickness of 30 μm. The thickness of each SU-8 photoresist layer was 10 μm. A mold with a highly gradient coupled microarray structure was made on a silicon wafer using ultraviolet lithography technology. The longitudinal configuration of the highly gradient coupled microarray structure was stepped, including three height regions, with a height region I of 30 μm, a height region II of 20 μm, and a height region III of 10 μm. The longitudinal configuration of the gradient The shape is sinusoidal, and then polydimethylsiloxane and isooctyl acrylate are mixed, tetrahydrofurfuryl acrylate, 2-naphthyl vinyl ketone, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide and curing agent are added, stirred at 3000rpm for 30min, allowed to stand in a dark place for 30min, and continued to stir evenly until the mixture is filled with fine and uniform bubbles, poured the mixture on the mold, placed in a vacuum box for 40min of degassing, reacted under ultraviolet light for 100s, and then placed in an oven at 85°C and heated for 90min. After the modified polydimethylsiloxane layer is completely cured, the modified polydimethylsiloxane layer is peeled off from the mold with a blade to obtain a modified polydimethylsiloxane layer with a size of 30mm×15mm×5mm.
[0040] In this embodiment, the polydimethylsiloxane and the curing agent are components of SYLGARD™ 184 Silicone Elastomer Kit of Dow, and the mass ratio of polydimethylsiloxane to the curing agent is 1:0.1; the volume ratio of polydimethylsiloxane to isooctyl acrylate is 1:1, the amount ratio of polydimethylsiloxane to tetrahydrofurfuryl acrylate is 1mL:0.4g, the amount ratio of polydimethylsiloxane to 2-naphthyl vinyl ketone is 1mL:0.3g, and the amount ratio of polydimethylsiloxane to phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide is 1mL:0.2g.
[0041] S2. Preparation of microfluidic chip, including: The glass was cut into a size of 50 mm × 30 mm × 1 mm. The modified polydimethylsiloxane layer obtained in step S1 and the surface of the glass layer facing the fluid area were first treated with air plasma. The modified polydimethylsiloxane layer obtained in step S1 and the glass layer were then bonded together. A chip inlet and a chip outlet with a diameter of 1 mm were then arranged at both ends to obtain a microfluidic chip.
[0042] S3. Capture of circulating tumor cells, same as in Example 1.
[0043] Embodiment 3: Except that in the preparation of the modified polydimethylsiloxane layer, the amount ratio of polydimethylsiloxane to tetrahydrofurfuryl acrylate was changed to 1 mL:0.1 g, other conditions were the same as those in Example 2.
[0044] Embodiment 4: Except that in the preparation of the modified polydimethylsiloxane layer, the amount ratio of polydimethylsiloxane to 2-naphthyl vinyl ketone is 1 mL:0.1 g, other conditions are the same as those in Example 2.
[0045] Embodiment 5: S1. Preparation of a modified polydimethylsiloxane layer, comprising: First, SU-8 photoresist was uniformly coated on a 4-inch silicon wafer to form a SU-8 photoresist layer with a thickness of 10 μm, and then spin-coated at a rotation speed of 3000 rpm for 1 minute, baked at 80°C for 1 hour, and then the silicon wafer was exposed to ultraviolet light for 3 minutes to cure the glue layer. This step was repeated twice to form a three-layer SU-8 photoresist structure with a total thickness of 30 μm. The thickness of each SU-8 photoresist layer was 10 μm. A mold with a highly gradient coupled microarray structure was made on a silicon wafer using ultraviolet lithography technology. The longitudinal configuration of the highly gradient coupled microarray structure was stepped, including three height regions, the size of height region I was 30 μm, the size of height region II was 20 μm, and the size of height region III was 10 μm. The longitudinal configuration of the gradient was sinusoidal. wavy, then mix polydimethylsiloxane and isooctyl acrylate, add tetrahydrofurfuryl acrylate, 2-naphthyl vinyl ketone, ethyl vinyl ether, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide and curing agent, stir at 3000rpm for 30min, let stand in a dark place for 30min, continue to stir evenly until the mixture is filled with fine and uniform bubbles, pour the mixture on the mold, place it in a vacuum box for 40min of degassing, react under ultraviolet light for 100s, and then put it in an oven at 85℃ and heat for 90min. After the modified polydimethylsiloxane layer is completely cured, use a blade to peel the modified polydimethylsiloxane layer from the mold to obtain an upper modified polydimethylsiloxane layer with a size of 30mm×15mm×5mm.
[0046] In this embodiment, the polydimethylsiloxane and the curing agent are components of SYLGARD™ 184 Silicone Elastomer Kit of Dow, and the mass ratio of polydimethylsiloxane to the curing agent is 1:0.1; the volume ratio of polydimethylsiloxane to isooctyl acrylate is 1:1, the amount ratio of polydimethylsiloxane to tetrahydrofurfuryl acrylate is 1mL:0.4g, the amount ratio of polydimethylsiloxane to 2-naphthyl vinyl ketone is 1mL:0.3g, the amount ratio of polydimethylsiloxane to ethyl vinyl ether is 1mL:0.2g, and the amount ratio of polydimethylsiloxane to phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide is 1mL:0.2g.
[0047] S2. Preparation of microfluidic chip, including: The glass was cut into a size of 50 mm × 30 mm × 1 mm. The modified polydimethylsiloxane layer obtained in step S1 and the surface of the glass layer facing the fluid area were first treated with air plasma. The modified polydimethylsiloxane layer obtained in step S1 and the glass layer were then bonded together. A chip inlet and a chip outlet with a diameter of 1 mm were then arranged at both ends to obtain a microfluidic chip.
[0048] S3. Capture of circulating tumor cells, same as in Example 1.
[0049] Embodiment 6: Except that in the preparation of the modified polydimethylsiloxane layer, the amount ratio of polydimethylsiloxane to ethyl vinyl ether was changed to 1 mL:0.1 g, other conditions were the same as those in Example 5.
[0050] Comparative Example 1: Except that 2-naphthyl vinyl ketone and ethyl vinyl ether were not added in the preparation of the modified polydimethylsiloxane layer, other conditions were the same as those in Example 2.
[0051] Comparative Example 2: Except that tetrahydrofurfuryl acrylate and ethyl vinyl ether were not added in the preparation of the modified polydimethylsiloxane layer, other conditions were the same as those in Example 2.
[0052] Comparative Example 3: Except that tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone were not added in the preparation of the modified polydimethylsiloxane layer, other conditions were the same as those in Example 2.
[0053] Experimental example: 1. Capture of circulating tumor cells Take 150 μL of MDA-MB-231 breast cancer cell sample with an average diameter of 15.5 μm. MDA-MB-231 breast cancer cells were purchased from the Experimental Center of Zhejiang Cancer Institute. Add 850 μL of pure DMEM culture medium. Use a pipette to fully blow the cell solution. After the cells are evenly dispersed, inject them into the chip through a syringe pump and a syringe at a flow rate of 1 mL / h. Observe in real time on a microscope and record the captured cell images.
[0054] Figure 1 The schematic diagram of the structure of the microfluidic chip prepared in Example 1 is as follows. The microfluidic chip includes a polydimethylsiloxane layer, a glass layer, a fluid region formed between the polydimethylsiloxane layer and the glass layer, and a chip inlet and a chip outlet with a diameter of 1 mm connected to the fluid region; Figure 2 A distribution map of height gradient regions in a microfluidic chip is prepared for Example 1. The polydimethylsiloxane layer of the microfluidic chip includes the entire height gradient coupled microarray structure, including microstructures of three height regions, wherein the height dimension of height region I is 30 μm, the height dimension of height region II is 20 μm, and the height dimension of height region III is 10 μm; Figure 3 The schematic diagram of the capture principle of the microfluidic chip prepared in Example 1 shows that after the fluid is injected into the microfluidic chip, circulating tumor cell clusters or circulating tumor cells larger than 10 μm will be blocked at each level of height gradient and cannot enter the height area of the next level, while blood cells smaller than 10 μm can pass normally and will be recovered from the outlet; Figure 4 This is an image of circulating tumor cells captured in Example 1, which shows that the method of Example 1 of the present invention can separate and capture circulating tumor cells on the gradient of the microfluidic chip according to the size of the circulating tumor cells.
[0055] 2. Material Characterization The infrared spectrum of the sample was measured by Fourier transform infrared spectrometer. In a dry environment, the attenuated total reflection accessory was placed in the optical path of the spectrometer, and the air background was scanned. The modified polydimethylsiloxane layer sample obtained in Example 5 was placed close to the crystal surface of the attenuated total reflection accessory to collect the infrared spectrum with a resolution of 4 cm -1 , scanned 32 times, the scanning range is 400-4000cm -1 .
[0056] Figure 5 This is the infrared spectrum of the modified polydimethylsiloxane layer obtained in Example 5, 2960 cm -1 and 2830cm -1 The absorption peak of CH appears near 1690 cm -1 There is a C=O absorption peak near 1630cm -1 The absorption peak of C=C appears near 1390cm -1The absorption peak of CO appears near 1030cm -1 The absorption peak of COC appears near 1257cm -1 and 785cm -1 The absorption peaks of Si-CH3 appear near 1085 and 863 cm -1 An absorption peak of Si-O-Si appears nearby.
[0057] 2. Determination of capture rate 150 μL of MDA-MB-231 breast cancer cell sample with an average diameter of 15.5 μm was taken. MDA-MB-231 breast cancer cells were purchased from the Experimental Center of Zhejiang Cancer Institute. 850 μL of pure DMEM culture medium was added. The cell solution was fully blown with a pipette. After the cells were evenly dispersed, they were injected into the microfluidic chip through a syringe pump and a syringe at a flow rate of 1 mL / h. The capture rate of breast cancer cells by the height gradient of the microfluidic chip was calculated. Capture rate (%) = number of captured breast cancer cells / total number of breast cancer cells × 100%. The measurement results are shown in Table 1.
[0058] Table 1 Capture rate (%)
[0059] The separation of circulating tumor cells based on physical properties has the problem of low capture rate, mainly because a very small number of circulating tumor cells are hidden in the background of a large number of blood cells, and their physical properties are quite different from those of blood cells but partially similar. Therefore, it is difficult to achieve efficient capture by relying solely on the size difference between circulating tumor cells and blood cells. To this end, the present invention constructs a microfluidic chip based on a height gradient, and optimizes its preparation process, introduces hydrophilic groups into the polydimethylsiloxane body, and forms a modified polydimethylsiloxane with an interpenetrating polymer network structure, thereby enhancing the dispersion effect of the microfluidic chip on biological particles, reducing the aggregation and adhesion of biological particles, separating circulating tumor cells from a large number of blood cell clusters, and preventing single cells from migrating, thereby improving the capture rate of circulating tumor cells by the microfluidic chip.
[0060] As can be seen from Table 1, compared with Example 1 and Example 2, the capture rate of Example 2 is higher than that of Example 1. The difference between Example 1 and Example 2 lies in the preparation of the microfluidic chip. Example 1 uses a polydimethylsiloxane layer to prepare the microfluidic chip; Example 2 first uses tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane to obtain a modified polydimethylsiloxane layer, and then uses the modified polydimethylsiloxane layer to prepare the microfluidic chip; this shows that the microfluidic chip obtained in Example 2 can improve the capture rate of circulating tumor cells.
[0061] Compared with Comparative Examples 1-2, Example 2 has a higher capture rate than Comparative Examples 1-2. The difference between Example 2 and Comparative Examples 1-2 lies in the preparation of the modified polydimethylsiloxane layer. Example 2 uses tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane at the same time to obtain a modified polydimethylsiloxane layer; Comparative Examples 1 and 2 only use one of tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane respectively; this shows that compared with using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone alone to modify polydimethylsiloxane, the modified polydimethylsiloxane layer obtained by synergistically using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane has a better effect on improving the capture rate of circulating tumor cells in the microfluidic chip.
[0062] Compared with Example 2 and Example 3-4, the capture rate of Example 2 is higher than that of Example 3-4. The difference between Example 2 and Example 3-4 is that in the preparation of the modified polydimethylsiloxane layer, the amounts of tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone used are different, which indicates that in the preparation of the microfluidic chip, the polydimethylsiloxane is modified with an appropriate amount of tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone, and the obtained modified polydimethylsiloxane layer helps to improve the capture rate of circulating tumor cells of the microfluidic chip.
[0063] Compared with Example 2 and Example 5, the capture rate of Example 5 is higher than that of Example 2. The difference between Example 2 and Example 5 lies in the preparation of the modified polydimethylsiloxane layer. In Example 5, ethyl vinyl ether is further used to modify polydimethylsiloxane on the basis of using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane; Compared with Comparative Example 3, the capture rate of Example 5 is higher than that of Comparative Example 3. The difference between Example 5 and Comparative Example 3 lies in the preparation of the modified polydimethylsiloxane layer. Comparative Example 3 only uses ethyl vinyl ether to modify polydimethylsiloxane; Compared with Example 5 and Example 6, the capture rate of Example 5 is higher than that of Example 6. This is because the amount of ethyl vinyl ether used in the preparation of the modified polydimethylsiloxane layer is different. This shows that on the basis of using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane, further using an appropriate amount of ethyl vinyl ether can further improve the capture rate of circulating tumor cells of the microfluidic chip.
[0064] 3. Determination of recovery rate 150 μL of MDA-MB-231 breast cancer cell sample with an average diameter of 15.5 μm was taken. MDA-MB-231 breast cancer cells were purchased from the Experimental Center of Zhejiang Cancer Institute. 850 μL of pure DMEM culture medium was added. The cell solution was fully blown with a pipette. After the cells were evenly dispersed, they were injected into the chip through a syringe pump and a syringe at a flow rate of 1 mL / h. After the breast cancer cells were captured on the gradient of the microfluidic chip, the inlet and outlet were swapped, and the captured breast cancer cells were recovered by reverse fluid flushing. The recovery rate of breast cancer cells was calculated. Recovery rate (%) = number of recovered breast cancer cells / total number of recovered biological particles × 100%. The measurement results are shown in Table 2.
[0065] Table 2 Recovery rate (%)
[0066] When separating circulating tumor cells based on physical properties, the problem of low recovery rate mainly stems from the fact that a very small number of circulating tumor cells are hidden in the background of a large number of blood cells, making it difficult to achieve high-purity recovery of circulating tumor cells. Therefore, the present invention optimizes the preparation process of the microfluidic chip, captures circulating tumor cells on the microfluidic chip, and achieves high-purity recovery through backwashing, thereby improving the recovery rate of circulating tumor cells.
[0067] As can be seen from Table 2, compared with Example 1 and Example 2, the recovery rate of Example 2 is higher than that of Example 1. The difference between Example 1 and Example 2 lies in the preparation of the microfluidic chip. Example 1 uses a polydimethylsiloxane layer to prepare the microfluidic chip; Example 2 first uses tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane to obtain a modified polydimethylsiloxane layer, and then uses the modified polydimethylsiloxane layer to prepare the microfluidic chip; This shows that the microfluidic chip obtained in Example 2 can improve the recovery rate of circulating tumor cells.
[0068] Compared with Comparative Examples 1-2, Example 2 has a higher recovery rate than Comparative Examples 1-2. The difference between Example 2 and Comparative Examples 1-2 lies in the preparation of the modified polydimethylsiloxane layer. Example 2 uses tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane at the same time to obtain a modified polydimethylsiloxane layer; Comparative Examples 1 and 2 only use one of tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane respectively; this shows that compared with using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone alone to modify polydimethylsiloxane, the modified polydimethylsiloxane layer obtained by synergistically using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane has a better effect on improving the recovery rate of circulating tumor cells in the microfluidic chip.
[0069] Compared with Example 2 and Example 3-4, the recovery rate of Example 2 is higher than that of Example 3-4. The difference between Example 2 and Example 3-4 is that in the preparation of the modified polydimethylsiloxane layer, the amounts of tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone used are different, which indicates that in the preparation of the microfluidic chip, the polydimethylsiloxane is modified using an appropriate amount of tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone, and the obtained modified polydimethylsiloxane layer helps to improve the recovery rate of circulating tumor cells in the microfluidic chip.
[0070] Compared with Example 2 and Example 5, the recovery rate of Example 5 is higher than that of Example 2. The difference between Example 2 and Example 5 lies in the preparation of the modified polydimethylsiloxane layer. In Example 5, ethyl vinyl ether is further used to modify polydimethylsiloxane on the basis of using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane; Compared with Comparative Example 3, the recovery rate of Example 5 is higher than that of Comparative Example 3. The difference between Example 5 and Comparative Example 3 lies in the preparation of the modified polydimethylsiloxane layer. Comparative Example 3 only uses ethyl vinyl ether to modify polydimethylsiloxane; Compared with Example 5 and Example 6, the recovery rate of Example 5 is higher than that of Example 6. This is because the amount of ethyl vinyl ether used in the preparation of the modified polydimethylsiloxane layer is different, which shows that on the basis of using tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone to modify polydimethylsiloxane, further using an appropriate amount of ethyl vinyl ether can further improve the recovery rate of circulating tumor cells in the microfluidic chip.
[0071] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.
[0072] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A microfluidic chip based on height gradient, comprising a polysiloxane layer, a glass layer, a fluid region formed between the polysiloxane layer and the glass layer, and a chip inlet and a chip outlet connected to the fluid region; the polysiloxane layer has a height gradient coupled microarray structure on its surface facing the fluid region; the height gradient coupled microarray structure has a stepped longitudinal configuration, including at least 3 height gradients, and the smallest height gradient size is 9-11 μm.
2. A microfluidic chip based on height gradient according to claim 1, characterized in that: The lateral configuration of the height gradient coupling microarray structure is wavy.
3. A microfluidic chip based on height gradient according to claim 1, characterized in that: The diameter of the chip inlet is 0.5-1.5 mm, and the diameter of the chip outlet is 0.5-1.5 mm.
4. A microfluidic chip based on height gradient according to claim 1, characterized in that: The polysiloxane layer includes a polydimethylsiloxane layer and / or a modified polydimethylsiloxane layer, wherein the modified polydimethylsiloxane layer has a naphthyl group and a carbonyl group.
5. A microfluidic chip based on height gradient according to claim 4, characterized in that: In the preparation of the modified polydimethylsiloxane, a highly gradient coupled microarray structure mold is firstly made on a silicon wafer, and then polydimethylsiloxane, isooctyl acrylate, a vinyl monomer substance, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide and a curing agent are mixed and poured on the mold for reaction to obtain a modified polydimethylsiloxane layer; the vinyl monomer substance at least includes tetrahydrofurfuryl acrylate and 2-naphthyl vinyl ketone.
6. A microfluidic chip based on height gradient according to claim 1, characterized in that: In the preparation of the microfluidic chip, the surfaces of the polysiloxane layer and the glass layer facing the fluid region are firstly treated with air plasma, and then the polysiloxane layer and the glass layer are bonded to obtain the microfluidic chip.
7. A method for capturing circulating tumor cells based on a microfluidic system based on a height gradient, comprising injecting a fluid into the microfluidic chip of claim 1 to capture circulating tumor cells, and recovering the circulating tumor cells by back flushing, wherein the fluid contains circulating tumor cells.
8. The method for capturing circulating tumor cells based on microfluidics with a height gradient according to claim 7, characterized in that: The amount of the fluid used is 0.5-2 mL.
9. The method for capturing circulating tumor cells based on microfluidics based on height gradient according to claim 7, characterized in that: The flow rate of the fluid is 0.2-2 mL / h.