Three-dimensional network chip and preparation method and application thereof

Abstract

The invention discloses a three-dimensional network chip and a preparation method and application thereof, the three-dimensional network chip comprises a polydimethylsiloxane film, the polydimethylsiloxane film has a three-dimensional network skeleton structure, and the surface of the three-dimensional network skeleton structure is loaded with gel composed of titanium carbide-Mxene and graphene oxide. According to the three-dimensional network chip, the three-dimensional network PDMS thin film serves as a chip substrate, the functionalized titanium carbide-Mxene / graphene oxide gel is loaded on the surface of the substrate, the functionalized three-dimensional network chip is constructed, and the three-dimensional network thin chip can be used for capturing circulating tumor cells. By utilizing the synergistic effect of the three-dimensional network structure of the chip and the Mxene / graphite oxide functionalized surface, the adhesion promoting effect on cancer cells is improved, the yield of circulating tumor cells is increased, and the capture efficiency can reach 90% or above within 1 h.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection materials, and in particular relates to a three-dimensional network chip and a preparation method and application thereof. Background Art

[0002] Circulating tumor cells (CTCs) refer to tumor cells that fall off from the primary tumor site and enter the blood circulation. Since circulating tumor cells appear in the early stages of cancer, using circulating tumor cells as biomarkers and conducting liquid biopsies is of great significance for early cancer detection. By monitoring and analyzing the number of circulating tumor cells in the blood, it has great application value in cancer staging, efficacy monitoring, and drug screening.

[0003] Traditional methods for capturing circulating tumor cells mainly include immunomagnetic bead adsorption, density gradient centrifugation, membrane filtration, and microfluidic chip methods. Some of these capture methods have problems such as complex operation, high cost, and difficulty in capture. In addition, due to the heterogeneity of tumors, the levels of tumor markers (e.g., EpCAM) expressed on the surface of tumor cells are inconsistent, so the capture method based on immunomagnetic bead technology may miss detection.

[0004] With the rapid development of nanomaterials, the high adhesion of tumor cells on specific structures and specific nanomaterial surfaces is used to capture circulating tumor cells, which is expected to achieve efficient capture of circulating tumor cells. Currently reported nanodevices include nanopillars, nanowires, and nanofibers. Carbon nanomaterials (such as carbon nanotubes and graphene) have also been used to help capture CTCs. However, common nanodevices require additional tumor-specific adhesion molecules (proteins for specific recognition, such as patent CN109821134A and patent CN118209727A). This type of capture method requires expensive tumor-specific antibodies or aptamers, etc., which are costly, the preparation steps are cumbersome, and the specific recognition molecules are easily inactivated. Therefore, the chip has a limited storage environment. In addition, not all CTCs express specific recognition molecules on their surfaces, which causes this method to have problems such as missed detection of CTCs and low detection rate. However, the existing nanochips for label-free capture of circulating tumor cells still have problems such as low capture efficiency, cumbersome preparation process, and environmental unfriendliness. For example, patent CN 113186164 A, the chip does not need to be modified with specific cancer cell recognition antibodies or nucleic acid aptamers, and can achieve the capture of different cancer cell types. It has the advantages of low cost and simple operation, but the capture efficiency is low. In addition, patent CN111225608A, the preparation method of the chip requires the use of harsh reaction conditions (high temperature, high pressure, flammable gas, volatile organic reagents) to obtain the nanostructured substrate material. In view of the above problems, it is necessary to develop a high capture efficiency, low cost and simple preparation of label-free capture of circulating tumor cells nanochip. Summary of the invention

[0005] In order to overcome the problems existing in the above-mentioned prior art, one of the purposes of the present invention is to provide a three-dimensional network chip. The second purpose of the present invention is to provide a method for preparing the above-mentioned three-dimensional network chip. The third purpose of the present invention is to provide an application of the above-mentioned three-dimensional network chip. The fourth purpose of the present invention is to provide a method for capturing circulating tumor cells.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A first aspect of the present invention provides a three-dimensional network chip, comprising a polydimethylsiloxane film having a three-dimensional network skeleton structure, the surface of which is loaded with a gel consisting of titanium carbide-Mxene and graphene oxide.

[0008] Titanium Carbide-Mxene(Ti 3 C 2 T x ) is a MXene material, which belongs to two-dimensional nanomaterials.

[0009] Preferably, the mass ratio of titanium carbide-Mxene to graphene oxide is (1-2):1.

[0010] The second aspect of the present invention provides a method for preparing the three-dimensional network chip according to the first aspect, comprising the following steps:

[0011] S1, etching the polydimethylsiloxane film with an etching solution to obtain a polydimethylsiloxane film having a three-dimensional network skeleton structure, i.e., a three-dimensional network film;

[0012] S2. The three-dimensional network film is loaded into a gel composed of titanium carbide-Mxene and graphene oxide to obtain a three-dimensional network chip.

[0013] Preferably, the method for preparing the polydimethylsiloxane film comprises the following steps: mixing polydimethylsiloxane and a curing agent into a prepolymer, coating the prepolymer on a substrate, and curing to form a film.

[0014] More preferably, the curing agent is selected from at least one of diethylenetriamine, triethylenetetramine and diethylaminopropylamine.

[0015] More preferably, the mass ratio of the polydimethylsiloxane to the curing agent is (8-12):1.

[0016] More preferably, the specific step of coating the prepolymer on the substrate comprises: placing the strip substrate in the prepolymer and dispersing the prepolymer to obtain a substrate loaded with the prepolymer.

[0017] More preferably, the curing temperature is 70-90° C. and the curing time is 2-5 hours.

[0018] Preferably, the etching process comprises: immersing the polydimethylsiloxane film in an etching solution.

[0019] Preferably, the etching solution is a nitric acid solution with a concentration of 6-8 mol / L.

[0020] Preferably, the etching temperature of the etching is 15-40°C.

[0021] Preferably, the etching time of the etching is 2-4 hours.

[0022] Preferably, the method for preparing the gel composed of titanium carbide-Mxene and graphene oxide comprises the following steps: mixing a titanium carbide-Mxene solution with a graphene oxide solution, reacting them, and preparing a gel.

[0023] Titanium carbide-MXene and graphene oxide can autonomously assemble into a three-dimensional gel structure through the interaction between two-dimensional layered structures.

[0024] More preferably, the concentration of the titanium carbide-Mxene solution is 5-20 mg / mL.

[0025] More preferably, the concentration of the graphite oxide solution is 5-20 mg / mL.

[0026] More preferably, the reaction time of the reaction is 3-6h.

[0027] Preferably, the loading time is 1-4 hours.

[0028] Preferably, step S2 is: immersing the three-dimensional network film in titanium carbide-Mxene / graphene oxide gel for loading, and then drying to obtain a three-dimensional network chip.

[0029] The three-dimensional network PDMS film has a high specific surface area based on physical adsorption.

[0030] More preferably, the drying temperature is 50-70°C.

[0031] The third aspect of the present invention provides an application of the three-dimensional network chip described in the first aspect in A) or B):

[0032] A) preparing a product for capturing circulating tumor cells;

[0033] B) Capture of circulating tumor cells.

[0034] Preferably, the circulating tumor cells are circulating human breast cancer cells.

[0035] More preferably, the circulating tumor cells are circulating tumor cells MCF-7.

[0036] The fourth aspect of the present invention provides a method for capturing circulating tumor cells, comprising the following steps: contacting the circulating tumor cells with the three-dimensional network chip as described in the first aspect.

[0037] The beneficial effects of the present invention are:

[0038] The present invention provides a three-dimensional network chip, which uses a three-dimensional network PDMS film as a chip substrate, and loads functionalized titanium carbide-Mxene / graphene oxide gel on the substrate surface to construct a functionalized three-dimensional network chip. The three-dimensional network thin chip can be used to capture circulating tumor cells. The three-dimensional network structure of the chip and the synergistic effect of the Mxene / graphene oxide functionalized surface are utilized to improve the adhesion promoting effect on cancer cells and the capture amount of circulating tumor cells, and the capture efficiency can reach more than 90% within 1 hour.

[0039] Specifically, compared with the prior art, the present invention has the following advantages:

[0040] (1) The three-dimensional network thin chip of the present invention can be directly used to capture various types of circulating tumor cells (regardless of the type of tumor cells, whether specific proteins are expressed on the surface of tumor cells, and the expression level) without any tumor biomarkers, and the application method is simple to operate. The prepared three-dimensional network chip has good biocompatibility and low toxicity, and the captured circulating tumor cells can be used for further culture and screening of anti-tumor drugs.

[0041] (2) The preparation method of the three-dimensional network chip of the present invention is simple and does not require harsh reaction conditions. The obtained titanium carbide-Mxene / graphene oxide gel is uniformly loaded on the three-dimensional network porous PDMS skeleton. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a scanning electron microscope image of a titanium carbide-Mxene / graphene oxide three-dimensional network chip; wherein, Figure a is a scanning electron microscope image, and Figure b is a high-magnification scanning electron microscope image of a part of Figure a;

[0043] Figure 2 This is a scanning diagram of the energy spectrum element distribution of the titanium carbide-Mxene / graphene oxide three-dimensional network chip;

[0044] Figure 3 This is a comparison chart of the cytotoxicity of titanium carbide-Mxene / graphene oxide three-dimensional network chips;

[0045] Figure 4 The time relationship diagram of the capture efficiency of MCF-7 cells captured by titanium carbide-Mxene / graphene oxide three-dimensional network chip;

[0046] Figure 5 Capture efficiency diagram of different numbers of MCF-7 cells captured for titanium carbide-Mxene / graphene oxide three-dimensional network chip application;

[0047] Figure 6 Linear graph of capture efficiency of different numbers of MCF-7 cells captured by titanium carbide-Mxene / graphene oxide three-dimensional network chip application;

[0048] Figure 7 Figure 2 is the capture efficiency diagram of different three-dimensional network chips. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below through specific examples. The raw materials used in the following examples, unless otherwise specified, can be obtained from conventional commercial sources or prepared and separated by simple synthesis; the processes used, unless otherwise specified, are conventional processes in the art.

[0050] The titanium carbide-Mxene used in the following examples and comparative examples was purchased from Nanjing Xianfeng Nanotechnology Co., Ltd., item number: 105119, serial number: XFK04-1.

[0051] Example 1

[0052] This embodiment provides a titanium carbide-Mxene / graphene oxide three-dimensional network chip, and the preparation method thereof is as follows:

[0053] 1. Preparation of titanium carbide-Mxene / graphene oxide gel

[0054] A titanium carbide-Mxene aqueous solution with a concentration of 10 mg / mL and a graphene oxide aqueous solution with a concentration of 10 mg / mL were respectively taken in glass bottles with a volume ratio of 3:2. The solutions were stirred at room temperature for 4 hours and then ultrasonicated for 20 minutes to obtain a titanium carbide-Mxene / graphene oxide gel.

[0055] 2. Preparation of three-dimensional network films

[0056] Weigh polydimethylsiloxane (PDMS) and curing agent in a weight ratio of 10:1 in a paper cup, stir evenly to obtain a PDMS prepolymer; put a strip of foam nickel cut into 4cm*0.8cm*1mm (length*width*thickness) into a 5mL EP tube, then fill the EP tube with PDMS prepolymer, then use a 4-degree low-temperature centrifuge, at a speed of 8000rpm, centrifuge for 5 minutes, take out the foam nickel with PDMS prepolymer loaded on the surface; then put it into a clean 5mL EP tube, continue to use a 4-degree low-temperature centrifuge, at a speed of 3000rpm, centrifuge for 3 minutes, remove excess PDMS, take out the foam nickel strip with PDMS prepolymer loaded on the surface, then dry it in an 80-degree oven for 3h to cure PDMS, and obtain a PDMS-cured foam nickel strip; finally, immerse it in a nitric acid solution with a concentration of 7mol / L, etch it at room temperature for 3h, and then wash it with pure water to obtain a three-dimensional network PDMS film.

[0057] 3. Preparation of titanium carbide-Mxene / graphene oxide three-dimensional network chip

[0058] The three-dimensional network PDMS film prepared above was immersed in the titanium carbide-Mxene / graphene oxide gel for 2 hours and then taken out, and then placed in a 60° C. oven for drying for 6 hours to obtain a titanium carbide-Mxene / graphene oxide three-dimensional network chip.

[0059] Comparative Example 1

[0060] This comparative example provides a graphene oxide three-dimensional network chip, and the preparation method thereof is as follows:

[0061] The three-dimensional network PDMS film prepared in Example 1 was immersed in a 10 mg / mL graphene oxide aqueous solution for 2 hours, then taken out and placed in an oven at 60 degrees for drying for 6 hours to obtain a graphene oxide three-dimensional network chip.

[0062] Comparative Example 2

[0063] This comparative example provides a titanium carbide-Mxene three-dimensional network chip, and its preparation method is as follows:

[0064] The three-dimensional network PDMS film prepared in Example 1 was immersed in a 10 mg / mL titanium carbide-Mxene aqueous solution for 2 hours, then taken out and placed in an oven at 60 degrees for drying for 6 hours to obtain a titanium carbide-Mxene three-dimensional network chip.

[0065] Material characterization

[0066] Figure 1Shown is a scanning electron microscope of a titanium carbide-Mxene / graphene oxide three-dimensional network chip. From the figure, it can be observed that the chip is a three-dimensional network porous structure, and the titanium carbide-Mxene / graphene oxide gel loaded on the surface of the chip skeleton presents an island-shaped raised rough structure.

[0067] Figure 2 Shown is the energy spectrum element distribution scan of the titanium carbide-Mxene / graphene oxide three-dimensional network chip. From the figure, it can be observed that carbon (C), oxygen (O), and titanium (Ti) elements are evenly distributed on the chip skeleton, proving that the titanium carbide-Mxene / graphene oxide gel is evenly loaded on the three-dimensional network porous PDMS skeleton.

[0068] Experimental analysis

[0069] Use a 6 mm diameter puncher to punch the titanium carbide-Mxene / graphene oxide three-dimensional network chip, titanium carbide-Mxene three-dimensional network chip and graphene oxide three-dimensional network chip prepared above to obtain a 6 mm diameter circular three-dimensional network chip for standby use.

[0070] 1. Cytotoxicity Characterization

[0071] Cell viability test: (1) Add 100 μL MCF-7 cells (cell density is 4*10 4 / mL) suspension to a 96-well plate (PBS is added to the edge wells). Set up three-dimensional network PDMS film holes, titanium carbide-Mxene / graphene oxide three-dimensional network chip holes, blank wells (with culture medium, no cells) and control wells (with cells and culture medium), with 5 replicate wells in each group. Culture in a cell culture incubator at 37 degrees and 5% carbon dioxide for 48 hours. (2) Add 10μL of CCK-8 solution to each well, and continue to incubate in a cell culture incubator at 37 degrees and 5% carbon dioxide for 1 hour. (3) Use a multifunctional microplate reader to test and determine the absorbance of each well at 450nm. Cell viability (%) = (measured value-blank value) / (control value-blank value)×100%.

[0072] Figure 3 The figure is a comparison chart of cytotoxicity of different substrates. It can be seen from the figure that the cell viability of the three-dimensional network PDMS substrate and the titanium carbide-Mxene / graphene oxide three-dimensional network substrate is slightly lower than that of the blank well plate substrate (control group), which are about 88% and 96% respectively. When the three-dimensional network PDMS substrate is loaded with titanium carbide-Mxene / graphene oxide gel, the substrate has higher cell viability. This shows that the titanium carbide-Mxene / graphene oxide three-dimensional network substrate has good biocompatibility and has little effect on cell viability.

[0073] 2. Capture efficiency experiment of MCF-7 cells

[0074] 100 μL of MCF-7 cell suspension with a cell density of 40,000 cells / mL was added to the 96-well plate containing the three-dimensional network chip of Example 1, and multiple experimental groups were incubated in a cell culture incubator for 15, 30, 45, 60, 90, and 120 min, respectively. At each time point, 3 groups of wells were repeated. Take out the 96-well plate, gently add phosphate buffer solution (1×PBS) along the wall of the well plate, soak for 2 minutes, and then flip the chip to allow the unattached cells to fall back into the suspension culture medium from the surface of the material by gravity. Then transfer the chip to a new blank 96-well plate, first fix it with 4% paraformaldehyde for 10 minutes, and wash it with PBS twice, each time for 2 minutes; then treat the chip with 0.25% Triton for 15 minutes, wash it with PBS twice, each time for 2 minutes, and finally use DAPI to stain the nuclei of CTCs on the chip for 5 minutes. Before using fluorescence microscopy for imaging observation, wash it with PBS twice, each time for 2 minutes. Using 10× objective lens, the imaging area is 1.144mm 2 (1.25 mm × 0.915 mm). Use a fluorescence camera to randomly take 9 fluorescence photos of the well plate cells, perform statistical calculations, and give the average.

[0075] The calculation method of capture efficiency is as follows: capture efficiency = capture amount / theoretical amount = (nA) / (Na), experimental cell density = number of image cells / image area = n / a; theoretical cell density = total number of cells / cell well plate area = N / A.

[0076] Figure 4 The capture efficiency time relationship diagram of MCF-7 cells captured by titanium carbide-MXene / graphene oxide three-dimensional network chip application. Figure 4 It can be seen that as the capture time increases, the capture efficiency of the chip for MCE-7 cells gradually increases. When the capture time is 1 hour, the capture efficiency reaches more than 90%, and as the capture time continues to increase, the capture efficiency does not show a significant improvement. Therefore, the optimal capture time is 1 hour.

[0077] 3. Capture efficiency experiment of different numbers of MCF-7 cells

[0078] 100 μL of MCF-7 cell suspension with cell density of (500, 1000, 2000, 5000, 10000, 40000) cells / mL was added to the 96-well plate containing the three-dimensional network chip, and three groups of duplicate wells were set up and incubated in the cell culture incubator for 1 hour. The 96-well plate was taken out, and phosphate buffer solution (1×PBS) was gently added along the wall of the well plate. After soaking for 2 minutes, the chip was flipped over to allow the unattached cells to fall back into the suspension culture medium from the surface of the material by gravity. Then the chip was transferred to a new blank 96-well plate, fixed with 4% paraformaldehyde for 10 minutes, and washed twice with PBS for 2 minutes each time; then the chip was treated with 0.25% Triton for 15 minutes, washed twice with PBS for 2 minutes each time, and finally the nuclei of CTCs on the chip were stained with DAPI for 5 minutes. Before using fluorescence microscopy imaging observation, it was washed twice with PBS for 2 minutes each time. Using 10× objective lens, the imaging area is 1.144mm 2 (1.25 mm × 0.915 mm). Use a fluorescence camera to randomly take 9 fluorescence photos of the well plate cells, perform statistical calculations, and obtain the average. The capture efficiency is calculated in the same way as above.

[0079] Figure 5 is the capture efficiency under different cell numbers, Figure 6 is the relationship between the number of cells and the number of captured cells. Figure 5 It can be seen that in the cases of 50, 100, 200, 500, 1000, and 4000 MCF-7 cells, the capture efficiency of the chip for MCF-7 cells is more than 90%. Figure 6 The number of cells captured in the chip gradually increases with the increase of the number of MCF-7 cells in the cell culture medium (DMEM), and the correlation is linear, indicating that the chip has good label-free capture ability for circulating tumor cells.

[0080] 4. Experiment on the capture efficiency of different numbers of MCF-7 cells using different three-dimensional network chips

[0081] Three-dimensional network PDMS film holes, titanium carbide-Mxene / graphene oxide three-dimensional network chip holes, graphene oxide three-dimensional network chip holes and titanium carbide-Mxene three-dimensional network chip holes were set in a 96-well cell culture plate, all of which were placed with a 6-mm diameter circular three-dimensional network chip (1 mm thick). 100 μL of a suspension culture of MCF-7 cells with a cell density of 40,000 cells / mL was injected into the 96-well cell culture plate with different three-dimensional network chips. After incubation in an incubator for 1 hour, phosphate buffer solution (1×PBS) was gently added along the wall of the well plate. After soaking for 2 minutes, the chip was flipped over to allow the unattached cells to fall back into the suspension culture medium from the material surface by gravity. The chip was then transferred to a new blank 96-well plate, fixed with 4% paraformaldehyde for 10 minutes, and washed twice with PBS for 2 minutes each time; the chip was then treated with 0.25% Triton X-ray for 15 minutes, washed twice with PBS for 2 minutes each time, and finally the nuclei of CTCs on the chip were stained with DAPI for 5 minutes. Before imaging with a fluorescent microscope, the chip was washed twice with PBS for 2 minutes each time. A 10× objective lens was used, and the imaging area was 1.144 mm 2 (1.25mm×0.915mm). Nine photos were taken randomly for each chip, and statistical calculations were performed to give the average. The calculation method for capture efficiency was the same as above.

[0082] from Figure 7 It can be seen that the titanium carbide-Mxene / graphene oxide three-dimensional network chip of Example 1 has the highest capture efficiency relative to the graphene oxide three-dimensional network chip of the single component (Comparative Example 1) and the titanium carbide-Mxene three-dimensional network chip (Comparative Example 2). The presumed reason is that the three-dimensional network chip loaded with the titanium carbide-Mxene / graphene oxide composite gel is more matched with the micro-nano topological structure of the MCF-7 cell surface, promoting the adhesion of MCF-7 cells and facilitating the enrichment and capture of cells.

[0083] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A three-dimensional network chip, characterized in that: The invention comprises a polydimethylsiloxane film having a three-dimensional network skeleton structure, and a gel composed of titanium carbide-Mxene and graphene oxide is loaded on the surface of the three-dimensional network skeleton structure.

2. The three-dimensional network chip according to claim 1, characterized in that: The mass ratio of the titanium carbide-Mxene to the graphene oxide is (1-2):

1.

3. The method for preparing the three-dimensional network chip according to claim 1 or 2, characterized in that: The steps include: S1, etching the polydimethylsiloxane film with an etching solution to obtain a polydimethylsiloxane film having a three-dimensional network skeleton structure, i.e., a three-dimensional network film; S2. The three-dimensional network film is loaded into a gel composed of titanium carbide-Mxene and graphene oxide to obtain a three-dimensional network chip.

4. The method for preparing a three-dimensional network chip according to claim 3, characterized in that: The method for preparing the polydimethylsiloxane film comprises the following steps: mixing polydimethylsiloxane and a curing agent to form a prepolymer, coating the prepolymer on a substrate, and curing the prepolymer to form a film.

5. The method for preparing a three-dimensional network chip according to claim 4, characterized in that: The curing temperature is 70-90° C. and the curing time is 2-5 hours.

6. The method for preparing a three-dimensional network chip according to claim 3, characterized in that: The etching solution is a nitric acid solution with a concentration of 6-8 mol / L; And / or, the etching time of the etching is 2-4h.

7. The method for preparing a three-dimensional network chip according to claim 3, characterized in that: The method for preparing the gel composed of titanium carbide-Mxene and graphene oxide comprises the following steps: mixing a titanium carbide-Mxene solution with a graphene oxide solution, reacting the mixture, and preparing the gel.

8. The method for preparing a three-dimensional network chip according to claim 3, characterized in that: The loading time is 1-4h.

9. Application of the three-dimensional network chip according to claim 1 or 2 in A) or B): A) preparing a product for capturing circulating tumor cells; B) Capture of circulating tumor cells.

10. A method for capturing circulating tumor cells, characterized in that: The method comprises the following steps: contacting circulating tumor cells with the three-dimensional network chip as claimed in claim 1 or 2.

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