Substrate preparation method and application for capturing and analyzing circulating tumor cells

Abstract

The present invention discloses a method for preparing a substrate for capturing and analyzing circulating tumor cells, including step S1, preparing a monolayer densely packed film of large-sized PS microspheres; step S2, lifting and transferring the monolayer densely packed film of large-sized PS microspheres prepared in step S1 to a PET film; step S3, preparing a micron cone array substrate; step S4, electron beam evaporation of a gold film; step S5, incubation of an aptamer on the substrate; the present invention also provides the application of the substrate prepared by the above-mentioned substrate preparation method for capturing and analyzing circulating tumor cells in a microfluidic chip. The periodically densely packed substrate of the present invention has a micro-nano heterogeneous microcone array with good SERS characteristics. After the aptamer is modified, it has a higher CTCs capture efficiency through the combination of the physical properties of the substrate and the affinity of the aptamer. The combination of the microfluidic chip and the SERS substrate improves the capture efficiency and provides a platform for capturing and detecting CTCs at the same time.

Description

Technical Field

[0001] The present invention relates to the field of chip technology, and in particular to a method for preparing a substrate for capturing and analyzing circulating tumor cells, and a specific application of a substrate prepared by the method for preparing a substrate for capturing and analyzing circulating tumor cells. Background Art

[0002] In recent years, the incidence and mortality of cancer have been increasing, and cancer has seriously endangered human life safety. The important causes of death of patients include cancer metastasis, which is often accompanied by several biological processes. Cancer cells fall off from the primary tissue of the tumor or the metastatic tissue, invade the lymphatic tissue or blood, spread to different tissues with the flow of blood, and serve as the cell source of cancer metastasis. These cancer cells separated from the primary tumor, invaded the blood and then entered the circulatory system are called circulating tumor cells (CTCs). CTCs contain a lot of valuable tumor information, and the number of CTCs is closely related to the stage, progression and treatment response of the disease. Clinical studies have shown that the survival rate of patients is negatively correlated with the number of CTCs in the blood. In particular, effective elimination of CTCs after adjuvant therapy can prolong the survival time of patients. However, there are many challenges in the capture and detection technology of CTCs, because unlike red blood cells, white blood cells and other blood cells, the content of CTCs is extremely small, which poses a great challenge to their separation and identification. Therefore, it is of great significance to design efficient and convenient devices for separating and analyzing CTCs.

[0003] Since the Indian physicist Raman discovered the Raman scattering phenomenon in the late 1920s, Fleischmann et al. discovered the surface enhanced Raman (SERS) phenomenon in 1974, which greatly compensated for the low sensitivity of Raman detection and quickly attracted the interest of physicists and chemists. People have been studying the internal principles of SERS enhancement. Based on these principles, people have been studying the substrate more and more deeply. People have found that in addition to the shape and size, the nano gaps between metals will also enhance the signal. Through these principles, SERS substrates are designed and manufactured, thus developing a series of substrates.

[0004] Microfluidic chips can also be called "laboratories on a chip". They can accurately control microfluidics and concentrate macroscopic laboratory operations and material detection on a chip. They have the advantages of portability, immediacy, and ease of operation, and have many applications in clinical testing, electrochemical experiments, cell experiments, and other fields. Since 1991, when A. Manz et al. transferred the electrophoresis separation experiment originally performed in capillaries to microfluidic chips, microfluidic chip technology has been increasingly valued by people and has also occupied an important position in the separation and detection of CTCs (circulating tumor cells).

[0005] CTCs were first discovered in the blood of cancer patients. They are similar to cancer cells in the tumor site, so they can be directly separated from the blood and used to study cancer treatment. At present, the main principles for separating and capturing CTCs can be roughly divided into two categories: the first is based on biological characteristics, based on specific biomarkers on the surface of CTCs; the second is based on the physical properties of CTCs, based on physical properties such as cell size, charge, density and elasticity. The detection technologies for CTCs include: fluorescence detection, electrochemical detection and Raman detection. Summary of the invention

[0006] The purpose of the present invention is to first solve the difficulty of densely packed monolayer arrangement of large-sized PS microspheres, and then use micromachining technology to prepare a micro-cone substrate with good SERS enhancement properties and CTCs capture performance, then deal with the problem of extremely low CTCs content and difficulty in separation, and finally provide a microfluidic platform for capture and detection.

[0007] In order to achieve the purpose of the invention, the present invention proposes the following technical solutions:

[0008] A method for preparing a substrate for capturing and analyzing circulating tumor cells comprises the following steps:

[0009] Step S1, preparation of a single-layer densely packed film of large-sized PS microspheres;

[0010] The glass slide is cleaned deeply, treated hydrophilically, and a PS microsphere ethanol mixture is prepared. A water film is added to the surface of the glass slide. The prepared PS microsphere ethanol mixture is added drop by drop to the edges of the glass slide. The microspheres are self-assembled into a single layer on the water surface. After natural air drying, a single-layer densely packed film of large-sized PS microspheres is obtained on the glass slide.

[0011] The step S1 comprises the following steps:

[0012] Step S101, using acetone, ethanol, concentrated sulfuric acid, hydrogen peroxide solution and deionized water in combination with ultrasound to deeply clean the glass sheet;

[0013] Step S102, performing ultrasonic hydrophilic treatment on the glass sheet using ammonia water, deionized water and hydrogen peroxide solution;

[0014] Step S103, preparing a PS microsphere ethanol mixture: taking 1 mL of a 20 μm PS microsphere mixture, centrifuging and taking out 600 μL of the supernatant, concentrating the PS microsphere mixture by 2.5 times, and mixing the concentrated PS microsphere mixture with ethanol at a ratio of 3:1 to prepare a PS microsphere ethanol mixture;

[0015] Step S104: drip a water film of about 2-3 mm on the surface of the dried glass sheet.

[0016] Step S105, adding the PS microsphere ethanol mixture prepared in step S103 dropwise to the corner of the glass sheet, and the microspheres self-assemble into a single layer on the surface of the water film;

[0017] Step S106, placing a dust-free cloth on a corner of the glass sheet to absorb excess water, and after natural air drying, a single-layer densely packed film of large-sized PS microspheres on the glass sheet is obtained.

[0018] Step S2, pulling and transferring the large-sized PS microsphere single-layer densely packed film prepared in step S1 onto the PET film;

[0019] The step S2 specifically includes:

[0020] Step S201, PET film processing;

[0021] Cut the PET film into appropriate size, clean it and put it into a plasma cleaning machine for 90 seconds; evenly coat the surface of the PET film with polyethylene glycol, and bake it at 150°C for 25 minutes; take out the PET film and let it cool to room temperature; rinse it with deionized water for 6-7 times to complete the hydrophilic treatment;

[0022] Step S202, insert the glass sheet obtained in step S1 into the ionized water surface at an angle of 45 degrees, and the single layer of balls will separate from the glass sheet and float on the water surface;

[0023] Step S203, adding a surfactant to reduce the surface tension of water and prevent the monolayer film from spreading;

[0024] Step S204, clamp the hydrophilic treated PET film with tweezers, immerse it in water at a position away from the PS film, and slowly move it to just below the single-layer film;

[0025] Step S205, slowly and evenly pulling the PET film out of the water at an angle of 45 degrees, thereby transferring the PS microsphere monolayer film suspended on the water surface to the surface of the PET film;

[0026] Step S206, after natural air drying, a single layer of densely packed large-sized PS microspheres on the PET film is obtained.

[0027] Step S3, preparation of micro-cone array substrate;

[0028] The PET film obtained in step S2 is placed in a reactive ion etcher to perform RIE etching to obtain a periodically densely packed micron cone array substrate;

[0029] The step S3 is specifically as follows: firstly, the air-dried PET film with the single-layer ball is placed in a reactive ion etcher for RIE etching, the etching power is adjusted to 270 W, and the O 2 The valve was opened and the air flow rate was adjusted to 20 sccm. The etching pressure was increased to 5 Pa. To prevent the machine from overloading, the etching was performed in four steps, each lasting 25 minutes with a 5-minute break in between. The total etching time was 100 minutes. Finally, a periodically densely packed micron-cone array substrate with a micro-nano hierarchical structure was obtained.

[0030] Step S4, electron beam evaporation of a gold film;

[0031] The prepared micro-cone array substrate is placed in a gold-plated chamber to evaporate a gold layer, thereby obtaining a SERS micro-cone array substrate with a uniform nano-gold film on the surface;

[0032] The prepared microcone array substrate was attached to a disc and placed in a gold-plated chamber. The vacuum was first evacuated and the gold plating speed was controlled to 0.03 nm.s -1 A film thickness meter was used to control the thickness of the evaporated gold layer on the substrate to about 100 nm, and a SERS microcone array substrate with a uniform nano-gold film on the surface was obtained.

[0033] Step S5, incubation of the aptamer on the substrate;

[0034] The substrate was placed in the specific aptamer solution, and the centrifuge tube was suspended on the edge of a vortex shaker. After slight oscillation was generated by collision with the edge of the vortex shaker, the substrate was taken out and rinsed with PBS solution for 2-3 times to obtain a SERS microcone substrate incubated with the aptamer.

[0035] Place the substrate into a plasma cleaning machine for treatment to improve the hydrophilicity of the substrate;

[0036] Close the valve, evacuate for about 90 s, then adjust the knob to "HI" and use ultraviolet light to perform oxygen plasma treatment for about 50 s;

[0037] Then slowly deflate the chamber to prevent the air from entering too quickly and blowing away the substrate;

[0038] Take out the substrate and put it into the 100 nmol.L prepared in advance. -1 In the Wy5a aptamer solution, in order to make the aptamer fully contact and modify the substrate, the centrifuge tube was suspended on the edge of the vortex shaker, and slight oscillation was generated by collision with the edge of the vortex shaker to avoid excessive oscillation of the centrifuge tube; after oscillating for 2 hours as above, the tube was taken out and rinsed with PBS solution for 2-3 times; finally, it was stored in a humid environment to obtain the SERS microcone substrate incubated with the aptamer.

[0039] The present invention also provides the use of a substrate prepared by the above-mentioned substrate preparation method for capturing and analyzing circulating tumor cells in a microfluidic chip.

[0040] The method for preparing a substrate for capturing and analyzing circulating tumor cells comprises the following steps:

[0041] The silicon positive template of the chip is prepared by photolithography technology, and the prepared PDMS (polydimethylsiloxane) mixture is poured into the culture dish fitted with the silicon positive template. After removing the bubbles, it is placed in an oven for drying. Then, the PDMS chip is peeled off with a knife and cut into single pieces to obtain a single-layer microfluidic chip.

[0042] The method also includes a further step of sealing the microfluidic chip and the substrate, specifically, ultrasonically cleaning the prepared single-layer microfluidic chip with ethanol, and then treating it with oxygen plasma with ultraviolet light for 90 s after drying. Then, the substrate that has completed the aptamer incubation is adhered to the chip chamber through the PDMS mixed liquid, and the two microfluidic chips are quickly attached to complete the assembly and sealing.

[0043] It also includes a further substrate characterization step, specifically including characterizing the morphology of the substrate through a scanning electron microscope; then characterizing the light absorption properties of the substrate through ultraviolet absorbance; and finally characterizing the SERS enhancement performance of the substrate through Raman reporter molecules.

[0044] Advantages and beneficial effects of the present invention:

[0045] 1. The periodically densely packed substrate with micro-nano heterogeneous microcone array has good SERS properties. After the aptamer is modified, it has a higher CTCs capture efficiency through the combination of the physical properties of the substrate and the affinity of the aptamer.

[0046] 2. The combination of microfluidic chip and SERS substrate improves the capture efficiency and provides a platform for capturing and detecting CTCs.

[0047] In this article, PS refers to polystyrene (PS), PET refers to polyethylene terephthalate (PET), and SERS refers to surface enhanced Raman effect (SERS). BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 Flowchart for processing and aptamer modification for microcone arrays;

[0050] Figure 2 is a scanning electron microscope image of a microcone array substrate;

[0051] Figure 3 It is the UV absorption diagram of different structural substrates;

[0052] Figure 4 Raman spectrum of MGITC measured for SERS substrate;

[0053] Figure 5 It is the uniformity detection and Raman Mapping diagram of SERS substrate;

[0054] Figure 6 Schematic diagram of microfluidic chip assembly;

[0055] Figure 7 Schematic diagram of microfluidic chip capture. ;

[0056] Figure 8 The SEM image of the captured cancer cells and the Raman mapping image of the bound detection antibody. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings.

[0058] Figure 1 This is a flow chart of the microcone array processing and aptamer modification used in the present invention. After obtaining a single layer of densely packed PS microspheres on a PET film, a periodically densely packed microcone substrate is prepared by using RIE etching and electron beam evaporation technology, and then the substrate is modified with an aptamer. The specific description is as follows:

[0059] Step S1, preparation of a single-layer densely packed film of large-sized PS microspheres;

[0060] The step S1 comprises the following steps:

[0061] Step S101, using acetone, ethanol, concentrated sulfuric acid, hydrogen peroxide solution and deionized water in combination with ultrasound to deeply clean the glass sheet;

[0062] The glass sheet used for laying the balls was cut into 5.0×5.0 cm square pieces, ultrasonically cleaned in acetone for 45 minutes, ultrasonically treated in ethanol for 30 minutes, and ultrasonically cleaned in deionized water for 30 minutes. After ultrasonic cleaning, it was placed in a solution of hydrogen peroxide and concentrated sulfuric acid in a ratio of 1:3 for 6 hours to deeply clean the impurities on the surface.

[0063] Step S102, performing ultrasonic hydrophilic treatment on the glass sheet using ammonia water, deionized water and hydrogen peroxide solution;

[0064] A hydrophilic treatment solution was prepared with ammonia water, hydrogen peroxide and deionized water in a ratio of 1:1:5. The glass slide was transferred into the hydrophilic treatment solution and ultrasonicated for 60 minutes.

[0065] Step S103, preparing a PS microsphere ethanol mixture: taking 1 mL of a 20 μm PS microsphere mixture, centrifuging and taking out 600 μL of the supernatant, concentrating the PS microsphere mixture by 2.5 times, and mixing the concentrated PS microsphere mixture with ethanol at a ratio of 3:1 to prepare a PS microsphere ethanol mixture;

[0066] Step S104, adding a water film of about 2-3 mm on the surface of the dried glass sheet;

[0067] Step S105, adding the PS microsphere ethanol mixture prepared in step S103 dropwise to the corner of the glass sheet, and the microspheres self-assemble into a single layer on the surface of the water film;

[0068] Step S106, placing a dust-free cloth on a corner of the glass sheet to absorb excess water, and after natural air drying, a single-layer densely packed film of large-sized PS microspheres on the glass sheet is obtained.

[0069] Step S2, pulling and transferring the large-sized PS microsphere single-layer densely packed film prepared in step S1 onto the PET film;

[0070] The step S2 specifically includes:

[0071] Step S201, PET film processing;

[0072] Cut the PET film into appropriate size, clean it and put it into a plasma cleaning machine for 90 seconds; evenly coat the surface of the PET film with polyethylene glycol, and bake it at 150°C for 25 minutes; take out the PET film and let it cool to room temperature; rinse it with deionized water for 6-7 times to complete the hydrophilic treatment;

[0073] Step S202, insert the glass sheet obtained in step S1 into the ionized water surface at an angle of 45 degrees, and the single layer of balls will separate from the glass sheet and float on the water surface;

[0074] Step S203, adding a surfactant to reduce the surface tension of water and prevent the monolayer film from spreading;

[0075] Step S204, clamp the hydrophilic treated PET film with tweezers, immerse it in water at a position away from the PS film, and slowly move it to just below the single-layer film;

[0076] Step S205, slowly and evenly pulling the PET film out of the water at an angle of 45 degrees, thereby transferring the PS microsphere monolayer film suspended on the water surface to the surface of the PET film;

[0077] Step S206, after natural air drying, a single layer of densely packed large-sized PS microspheres on the PET film is obtained.

[0078] Step S3, preparation of micro-cone array substrate;

[0079] The PET film obtained in step S2 is placed in a reactive ion etcher to perform RIE etching to obtain a periodically densely packed micron cone array substrate;

[0080] The step S3 is specifically as follows: firstly, the air-dried PET film with the single-layer ball is placed in a reactive ion etcher for RIE etching, the etching power is adjusted to 270 W, and the O 2 The valve was opened and the air flow rate was adjusted to 20 sccm. The etching pressure was increased to 5 Pa. To prevent the machine from overloading, the etching was performed in four steps, each lasting 25 minutes with a 5-minute break in between. The total etching time was 100 minutes. Finally, a periodically densely packed micron-cone array substrate with a micro-nano hierarchical structure was obtained.

[0081] Step S4, electron beam evaporation of a gold film;

[0082] The prepared micro-cone array substrate is placed in a gold-plated chamber to evaporate a gold layer, thereby obtaining a SERS micro-cone array substrate with a uniform nano-gold film on the surface;

[0083] The prepared microcone array substrate was attached to a disc and placed in a gold-plated chamber. The vacuum was first evacuated and the gold plating speed was controlled to 0.03 nm.s -1 A film thickness meter was used to control the thickness of the evaporated gold layer on the substrate to about 100 nm, and a SERS microcone array substrate with a uniform nano-gold film on the surface was obtained.

[0084] Step S5, incubation of the aptamer on the substrate;

[0085] Place the substrate into a plasma cleaning machine for treatment to improve the hydrophilicity of the substrate;

[0086] Close the valve, evacuate for about 90 s, then adjust the knob to "HI" and use ultraviolet light to perform oxygen plasma treatment for about 50 s;

[0087] Then slowly deflate the chamber to prevent the air from entering too quickly and blowing away the substrate;

[0088] Take out the substrate and put it into the 100 nmol.L prepared in advance. -1In the Wy5a aptamer solution, in order to make the aptamer fully contact and modify the substrate, the centrifuge tube was suspended on the edge of the vortex shaker, and slight oscillation was generated by collision with the edge of the vortex shaker to avoid excessive oscillation of the centrifuge tube; after oscillating for 2 hours as above, the tube was taken out and rinsed with PBS solution for 2-3 times; finally, it was stored in a humid environment to obtain the SERS microcone substrate incubated with the aptamer.

[0089] Figure 2 This is the morphology of the microcone array substrate prepared by the present invention. The average height of the microcone is about 30.81 μm, the average diameter of the microcone bottom is about 16.16 μm, and the gap between the microcones and the bottom of the microcones is about 7.55 μm. Due to the close periodic arrangement of the microspheres, the top spacing of the microcones after etching is about 20 μm, and the aspect ratio is about 1.91. At the same time, the sidewalls of the microcones also have a large number of different wrinkles and filamentous grooves, forming micro-nano heterogeneous structures. These structures not only improve the SERS enhancement performance of the substrate, but also play a vital role in the incubation of aptamers and the adhesion of cancer cells.

[0090] Figure 3 The following are the UV absorption graphs of substrates with different structures, which are the UV absorbance test results of the original PET film, the PET film coated with a 30 nm thick gold film, the substrate etched with a micro-cone array, the micro-cone substrate coated with a 30 nm thick gold film, and the micro-cone substrate coated with a 100 nm thick gold film. It can be seen from the figure that the light absorption performance of the PET film coated with a gold film is significantly reduced. This is because the relatively flat morphology enhances the ability to reflect light after gold coating, resulting in a decrease in light absorption performance. The light absorption performance of the substrate etched with a micro-cone array is greatly improved. This is because the wrinkled structure of the micro-cones and the side walls provides a longer light path and can absorb light more effectively. The light absorption performance of the micro-cone substrate after gold coating is further improved. It may be that the gold film on the surface and the micro-nano heterogeneous structure form a light trap, which allows light to be continuously refracted in the structure, effectively retaining the scattered and incident light, and improving the light absorption performance, which further proves that the substrate has a good SERS enhancement effect.

[0091] Figure 4 The Raman spectra of MGITC measured by SERS substrates are shown in Figure 2. The six groups of gold-plated substrates were placed in a 10% concentration -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 mol.L -1The substrate was immersed in MGITC solution for three hours, then taken out and air-dried for Raman detection. A laser with a wavelength of 633 nm was selected as the excitation wavelength, the laser power was selected as 25%, the integration time was 10 s, and the number of integrations was 3. The signal was denoised and baselined using LabSpec6.0 software. The result of each spectrum line is the average spectrum line of five different sampling points at the concentration. From the figure, it can be observed that the signal of the MGITC molecule is at 1614 cm - 1 The standard characteristic peak at , the signal peak gradually decreases as the concentration decreases.

[0092] Figure 5 The uniformity detection and Raman mapping of the SERS substrate are shown in Figure 1. The substrate is immersed in a 10 -7 mol.L -1 The samples were placed in MGITC solution for 3 hours. After drying, 20 random points were selected to measure the Raman signals. The combined Raman waterfall diagram is shown in Figure 2. Figure 5 (a). The relative standard deviation (RSD) calculated based on the peak value in the statistics is 9.22%, as shown in Figure 5 (b). The substrate is placed at a concentration of 10 -5 mol.L -1 The samples were placed in a Rhodamine 6G (R6G) solution for 2 hours, and then dried for Raman mapping detection. The Raman detection parameters were adjusted based on one of the characteristic peaks of R6G at 609 cm -1 , we set the scanning detection range to 590 cm -1 Up to 640cm -1 , a laser with a wavelength of 633 nm was selected as the excitation wavelength, the laser power was selected as 50%, the integration time was 1 s, the number of integrations was 1, the step size was set to 2 μm, and 50x50 unit points were collected, such as Figure 5 (c) It can be seen from the figure that the substrate has good uniformity.

[0093] The present invention also provides the use of a substrate prepared by the above-mentioned substrate preparation method for capturing and analyzing circulating tumor cells in a microfluidic chip.

[0094] Figure 6 is a schematic diagram of the microfluidic chip assembly, such as Figure 6 As shown, the modified aptamer substrate is attached to the chamber of the microchannel chip, and the microchannel chip and the herringbone channel chip are aligned and sealed, and finally the desired microfluidic chip is obtained.

[0095] The specific steps include:

[0096] First, use photolithography to prepare the positive template of the microchannel and herringbone channel chip, and attach it to the bottom of the culture dish. On the cavity of the pattern on the surface of the microchannel template, place a specially finely processed stainless steel gasket, and remove the gasket when peeling the PDMS chip later. It is mainly used to increase the height of the chip cavity so that the microcone base can be well embedded and the herringbone channel template does not move. Prepare the polydimethylsiloxane (PDMS) prepolymer and curing agent in a ratio of 10:1 to form a PDMS mixture, and pour it into the culture dish with the silicon positive template attached. Put it on the heating plate and adjust it to 75℃ for about 10 minutes. Adjust the position of the gasket so that it fits the chamber area tightly. At the same time, some bubbles will appear during the heating step. Use an ear suction bulb to blow them away in time to ensure that there are no bubbles in the culture dish. After dealing with the bubbles, put the culture dish flat in the oven, adjust the temperature to 75℃ and bake for about two hours to completely solidify the PDMS mixture, and finally turn it into a transparent flexible solid. Use a scalpel to peel off the solidified PDMS, and then use a utility knife to cut the edge of the chip, and use a hole puncher to punch a hole straight down at the designated position of the chip for the subsequent solution to enter and exit. This completes the preparation of the microfluidic chip.

[0097] The method also includes a further step of sealing the microfluidic chip with the substrate, specifically, ultrasonically cleaning the prepared microchannel and herringbone channel microfluidic chips with ethanol for 20 minutes, placing them in a plasma cleaner PDC-32G-2 after drying, first extracting the air in the internal chamber of the plasma cleaner for about 90 seconds, then adjusting the knob to "HI", and using ultraviolet light for oxygen plasma treatment for 90 seconds, and then adhering the substrate that has completed the aptamer incubation to the microchannel chip chamber through the PDMS mixed liquid, aligning the single-layer channel chip and the herringbone channel chip to a good position and quickly fitting them for sealing, and then using a suitable tool to press the chip surface to fully seal the chip to complete the chip assembly.

[0098] It also includes further substrate characterization steps, specifically including first characterizing the morphology of the substrate by scanning electron microscopy. Then, the light absorption properties of the substrate are characterized by using UV absorbance, and the substrate is placed in a UV-visible-near infrared spectrophotometer to compare the light absorption properties of different substrates; the SERS enhancement performance of the substrate is characterized by Raman reporter molecules, and the substrate is immersed in different concentrations of malachite green isothiocyanate (MGITC) and detected using the LabRam HREvolution laser confocal Raman spectroscopy system.

[0099] Figure 7 This is a schematic diagram of microfluidic chip capture. The patient's blood is introduced through the second inlet. After the cancer cells are captured, the detection antibody probe is introduced through the first inlet. The outlet is the waste liquid collection area.

[0100] Figure 8 It is a SEM image of the captured cancer cells and a Raman Mapping image of the combined detection antibody. Figure 8 As shown in (a), cancer cells bind to the aptamer on the cone, and their pseudopodia contact the cone tip and are captured by the cone tip. Then, gold nanoparticles, detection antibodies and Raman reporter molecules are combined, and the combined Raman probe will bind to the corresponding antigen on CTCs, thereby being used for Raman analysis and verification. The specific steps are as follows:

[0101] Take 1 mL of gold colloid and add 5 μL of 10-5 mol.L -1 The MGITC solution (Raman reporter molecule) was suspended on the edge of a vortex shaker for 30 minutes, and then a 0.1 mol.L -1 100 μL of borate buffer and 10 μL of 100 ng.ml -1 of detection antibody solution, mix thoroughly and shake for 2 hours. Slowly and evenly add 20 μL of 10% BSA by mass, and then suspend and shake for 30 minutes. After completion, put it in the upper layer of the refrigerator at 4°C and let it stand for one hour. Set the centrifuge parameters to 5200 rpm and centrifuge for 15 minutes. After the end, use a pipette to aspirate the supernatant, that is, remove the mixture of unbound detection antibodies, Raman reporter molecules or gold balls. Finally, add 0.25 mL of deionized water to the precipitate and shake it thoroughly.

[0102] Adjust the parameters of Raman detection to perform Raman Mapping detection. According to one of the characteristic peaks of MGITC, 1614 cm -1 , the detection range of our scan is set to 1590 cm -1 To 1640 cm -1 , collect 6x6 unit points, and set the step size to 4 μm. The brightness of the color in the Mapping diagram represents the strength of the signal. The brighter the color, the stronger the signal strength and the higher the peak value. Figure 8 It can be seen that the periphery is relatively dark, and the brighter it is towards the center, and the image approximately presents an area with a diameter of about 15-25 μm. Since the signal comes from the Raman probe bound to the detection antibody, and the detection antibody only specifically binds to CTCs, it proves that the captured ones are CTCs, once again verifying the role of the substrate in capturing cancer cells.

[0103] As described above, all equivalent changes and modifications made to the spectral data contained in the patent application scope of the present invention are covered by the scope of the claims of the present invention.

Claims

1. A method for preparing a substrate for capturing and analyzing circulating tumor cells, characterized in that: The following steps are involved: Step S1, preparation of a single-layer densely packed film of large-sized PS microspheres; The glass slide is cleaned deeply, treated hydrophilically, and a PS microsphere ethanol mixture is prepared. A water film is added to the surface of the glass slide. The prepared PS microsphere ethanol mixture is added drop by drop to the edges of the glass slide. The microspheres are self-assembled into a single layer on the water surface. After natural air drying, a single-layer densely packed film of large-sized PS microspheres is obtained on the glass slide. Step S2, pulling and transferring the large-sized PS microsphere single-layer densely packed film prepared in step S1 onto the PET film; Step S3, preparation of micro-cone array substrate; The PET film obtained in step S2 is placed in a reactive ion etcher to perform RIE etching to obtain a periodically densely packed micron cone array substrate; Step S4, electron beam evaporation of a gold film; The prepared micro-cone array substrate is placed in a gold-plated chamber to evaporate a gold layer, thereby obtaining a SERS micro-cone array substrate with a uniform nano-gold film on the surface; Step S5, incubation of the aptamer on the substrate; The substrate was placed in the specific aptamer solution, and the centrifuge tube was suspended on the edge of a vortex shaker. After slight oscillation was generated by collision with the edge of the vortex shaker, the substrate was taken out and rinsed with PBS solution for 2-3 times to obtain a SERS microcone substrate incubated with the aptamer.

2. The method for preparing a substrate for capturing and analyzing circulating tumor cells according to claim 1, characterized in that: The step S1 comprises the following steps: Step S101, using acetone, ethanol, concentrated sulfuric acid, hydrogen peroxide solution and deionized water in combination with ultrasound to deeply clean the glass sheet; Step S102, performing ultrasonic hydrophilic treatment on the glass sheet using ammonia water, deionized water and hydrogen peroxide solution; Step S103, preparing a PS microsphere ethanol mixture: taking 1 mL of a 20 μm PS microsphere mixture, centrifuging and taking out 600 μL of the supernatant, concentrating the PS microsphere mixture by 2.5 times, and mixing the concentrated PS microsphere mixture with ethanol at a ratio of 3:1 to prepare a PS microsphere ethanol mixture; Step S104: drip a water film of about 2-3 mm on the surface of the dried glass sheet. Step S105, adding the PS microsphere ethanol mixture prepared in step S103 dropwise to the corner of the glass sheet, and the microspheres self-assemble into a single layer on the surface of the water film; Step S106, placing a dust-free cloth on a corner of the glass sheet to absorb excess water, and after natural air drying, a single-layer densely packed film of large-sized PS microspheres on the glass sheet is obtained.

3. The method for preparing a substrate for capturing and analyzing circulating tumor cells according to claim 1, characterized in that: The step S2 specifically includes: Step S201, PET film processing; The PET film was cut into suitable sizes, cleaned and placed in a plasma cleaning machine for 90 seconds; Evenly coat the surface of the PET film with polyethylene glycol and bake at 150°C for 25 minutes; Take out the PET film and let it air to room temperature; Rinse with deionized water 6-7 times to complete the hydrophilic treatment; Step S202, insert the glass sheet obtained in step S1 into the ionized water surface at an angle of 45 degrees, and the single layer of balls will separate from the glass sheet and float on the water surface; Step S203, adding a surfactant to reduce the surface tension of water and prevent the monolayer film from spreading; Step S204, clamp the hydrophilic treated PET film with tweezers, immerse it in water at a position away from the PS film, and slowly move it to just below the single-layer film; Step S205, slowly and evenly pulling the PET film out of the water at an angle of 45 degrees, thereby transferring the PS microsphere monolayer film suspended on the water surface to the surface of the PET film; Step S206, after natural air drying, a single layer of densely packed large-sized PS microspheres on the PET film is obtained.

4. The method for preparing a substrate for capturing and analyzing circulating tumor cells according to claim 1, characterized in that: The step S3 is specifically as follows: first, the air-dried PET film with a single layer of balls is placed in a reactive ion etcher for RIE etching, the etching power is adjusted to 270 W, the O2 valve is opened, the air flow rate is adjusted to 20 sccm, the etching pressure is increased to 5 Pa, and to prevent the machine from overloading, the etching is performed in four times, each for 25 minutes, with a 5-minute pause in between, and a total etching time of 100 minutes; finally, a micron cone array substrate with a periodically densely packed arrangement and a micro-nano hierarchical structure is obtained.

5. The method for preparing a substrate for capturing and analyzing circulating tumor cells according to claim 1, characterized in that: The step S4 is specifically as follows: The prepared microcone array substrate was attached to a disc and placed in a gold-plated chamber. The vacuum was first evacuated and the gold plating speed was controlled to 0.03 nm.s -1 A film thickness meter was used to control the thickness of the evaporated gold layer on the substrate to about 100 nm, and a SERS microcone array substrate with a uniform nano-gold film on the surface was obtained.

6. The method for preparing a substrate for capturing and analyzing circulating tumor cells according to claim 1, characterized in that: The step S5 is specifically as follows: Place the substrate into a plasma cleaning machine for treatment to improve the hydrophilicity of the substrate; Close the valve, evacuate the chamber for about 90 seconds, then adjust the knob to "HI" and use ultraviolet light to perform oxygen plasma treatment for about 50 seconds; Then slowly deflate the chamber to prevent the air from entering too quickly and blowing away the substrate; Take out the substrate and put it into the 100 nmol.L prepared in advance. -1 In the Wy5a aptamer solution, in order to make the aptamer fully contact and modify the substrate, the centrifuge tube was suspended on the edge of the vortex shaker, and slight oscillation was generated by collision with the edge of the vortex shaker to avoid excessive oscillation of the centrifuge tube; after oscillating for 2 hours as above, the tube was taken out and rinsed with PBS solution for 2-3 times; finally, it was stored in a humid environment to obtain the SERS microcone substrate incubated with the aptamer.

7. Use of a substrate prepared by the method for preparing a substrate for capturing and analyzing circulating tumor cells as claimed in any one of claims 1 to 5 in a microfluidic chip.

8. Use of the substrate for capturing and analyzing circulating tumor cells according to claim 6 in a microfluidic chip, characterized in that: The method for preparing the microfluidic chip comprises the following steps: The silicon positive template of the chip is prepared by photolithography technology, and the prepared PDMS (polydimethylsiloxane) mixture is poured into the culture dish fitted with the silicon positive template. After removing the bubbles, it is placed in an oven for drying. Then, the PDMS chip is peeled off with a knife and cut into single pieces to obtain a single-layer microfluidic chip.

9. Use of the substrate for capturing and analyzing circulating tumor cells according to claim 7 in a microfluidic chip, characterized in that: The method also includes a further step of sealing the microfluidic chip and the substrate, specifically, ultrasonically cleaning the prepared single-layer microfluidic chip with ethanol, and then treating it with oxygen plasma with ultraviolet light for 90 s after drying. Then, the substrate that has completed the aptamer incubation is adhered to the chip chamber through the PDMS mixed liquid, and the two microfluidic chips are quickly attached to complete the assembly and sealing.

10. Use of the substrate for capturing and analyzing circulating tumor cells according to claim 8 in a microfluidic chip, characterized in that: It also includes a further substrate characterization step, specifically including characterizing the morphology of the substrate through a scanning electron microscope; then characterizing the light absorption properties of the substrate through ultraviolet absorbance; and finally characterizing the SERS enhancement performance of the substrate through Raman reporter molecules.

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