Microfluidic chip embedded with su-8 gel substrate for ctc capture and analysis

Abstract

The application discloses a kind of microfluidic chips for CTCs capture and analysis embedded SU-8 glue base, comprising the following steps: preparation of inverted-pyramid pit array silicon mother film;Secondary inverted mould pressurization printing preparation pyramid microcone array SU-8 glue base;Micro-nano processing preparation SU-8 glue base surface rough nano topography;E-beam evaporation gold film;Modification aptamer on SU-8 glue base;Preparation of microfluidic chip;Sealing of embedded base type chip;Characterization of SU-8 glue base;The pyramid microcone array SU-8 glue base of the application has good SERS characteristics, and the microcone array with micro-nano heterogeneous structure has higher CTCs capture efficiency through the physical properties of the base and the affinity of aptamer after modification. The combination of microfluidic chip and SERS base improves the capture efficiency and provides a platform for simultaneous capture and SERS detection of CTCs.

Description

Technical Field

[0001] This invention relates to the field of chip technology, and more specifically, to a microfluidic chip embedded in an SU-8 adhesive substrate for the capture and analysis of CTCs. Background Technology

[0002] In recent years, malignant tumors have shown signs of affecting younger people. It is estimated that by 2030, there will be approximately 20 million new cancer cases and 13 million cancer-related deaths worldwide. For cancer patients, early and successful diagnosis can prolong their lives. However, current diagnostic methods and treatments heavily rely on molecular-level identification. Furthermore, cancer cells can produce gene mutations and abnormal protein expression, making it difficult to effectively address the interference of false negatives and false positives in clinical results. Circulating tumor cells (CTCs), rare tumor cells that detach from the primary tumor and enter the circulatory system, are closely related to tumor metastasis. CTCs are also considered an important detection tool for early tumor diagnosis, prognostic assessment, and recurrence identification. Therefore, the capture, analysis, and detection of CTCs are an effective supplement to cancer detection. CTCs also carry a large number of tumor lesion characteristics, which are of great significance for clinical guidance. CTC detection information also provides important assistance in assessing the condition of patients with advanced cancer.

[0003] Since the discovery of Raman scattering by Indian physicist Raman in the late 1920s, and the subsequent discovery of surface-enhanced Raman scattering (SERS) by Fleischmann et al. in 1974, the discovery of SERS has greatly compensated for the low sensitivity of Raman detection. Its accepted enhancement mechanisms are electromagnetic enhancement and chemical enhancement. Based on these two enhancement principles, researchers have developed a series of SERS substrates.

[0004] Microfluidic chips are a scientific technology that controls fluids in a micrometer-scale space. This technology has the ability to condense the basic functions of biological and chemical laboratories onto a chip of less than a few square centimeters, hence the name "lab-on-a-chip." Most mainstream microfluidic chips currently consist of a network of microchannels, with a controllable fluid flowing through the entire chip to achieve various functions required for routine chemical or biological experiments. Their fundamental characteristic and greatest advantage lies in the ingenious combination and large-scale integration of various microstructural units on a tiny, controllable platform to accomplish functions such as detection and analysis.

[0005] Cytokinesous tumor cells (CTCs) are found in the peripheral blood of cancer patients and are rare tumor cells that detach from the primary tumor and enter the circulatory system. They are important detection tools for early tumor diagnosis, prognostic assessment, and recurrence identification. Currently, the main principles used for CTC isolation and capture can be broadly divided into two categories: the first is based on biological characteristics, relying on specific biomarkers on the surface of CTCs; the second is based on the physical characteristics of CTCs, relying on physical properties such as cell size, charge, density, and elasticity. CTC detection techniques include fluorescence detection, electrochemical detection, and Raman spectroscopy.

[0006] The inventor's prior patent application, application number 202210556477.2, application date: 2022-05-19, entitled "Method and Application of Substrate Preparation for Circulating Tumor Cell Capture and Analysis," discloses a method for preparing a substrate for capturing and analyzing circulating tumor cells (CTCs). The method includes steps S1, preparing a monolayer densely packed film of large-sized PS microspheres; step S2, transferring the monolayer densely packed film of large-sized PS microspheres obtained in step S1 onto a PET film; step S3, preparing a micron-cone array substrate; step S4, electron beam evaporation of a gold film; and step S5, aptamer incubation on the substrate. This invention also provides the application of the substrate prepared by the above-mentioned method for capturing and analyzing CTCs in a microfluidic chip. The periodically densely packed substrate of this invention possesses excellent SERS properties due to its micro / nano heterogeneous microcone array. After aptamer modification, the substrate exhibits high CTCs capture efficiency through the combination of the physical properties of the substrate and the affinity of the aptamer. The combination of microfluidic chips and SERS substrates improves capture efficiency and provides a platform that can simultaneously capture and detect CTCs. Summary of the Invention

[0007] The purpose of this invention is to solve the problem of low efficiency in fluid capture of circulating tumor cells. It uses deterministic lateral displacement to increase the concentration of CTCs, and then utilizes large-scale microcones with nanostructures on their surfaces to increase the probability of collision with circulating tumor cells at the microscale. At the same time, the nano-roughness of the surface induces cells to extend pseudopodia and provides hot spots to enhance the SERS signal. Finally, the two are combined to provide a microfluidic platform with both capture and detection capabilities.

[0008] To achieve the objective of this invention, the following technical solution is proposed:

[0009] A microfluidic chip embedded in an SU-8 adhesive substrate for CTC capture and analysis, the fabrication of which includes the following steps:

[0010] Step S1, preparation of the inverted pyramid pit array silicon mother film;

[0011] Step S10: Photolithography is performed on the silicon wafer with silicon dioxide (SiO2) oxide film to obtain square windows arranged in staggered columns;

[0012] Step S11: Use hydrofluoric acid to remove the SiO2 film exposed inside the window;

[0013] In step S12, the pattern is transferred to the SiO2 film layer;

[0014] Step S13: Add isopropanol (IPA) and deionized water to potassium hydroxide (KOH) solution to obtain KOH anisotropic etching solution;

[0015] Step S14: Place the silicon wafer with the pre-cut window inside the magnetic stirrer and etch it to obtain the inverted pyramid pit silicon master mold.

[0016] The specific parameters in step S1 are as follows: Photolithography is performed on a silicon wafer with a 300 nm thick silicon dioxide (SiO2) oxide film to obtain square windows with a side length of 160 μm, a spacing of 30 μm, and staggered arrangement in each column. The exposed SiO2 film within the windows is removed using hydrofluoric acid, and the pattern is transferred to the SiO2 film layer. 6 mol·L⁻¹ -1 10.5 ml of potassium hydroxide (KOH) solution was mixed with 1 ml of isopropanol (IPA) and 1 ml of deionized water to obtain a 5 mol / L solution. -1 KOH anisotropic etching solution was used. The temperature of the thermostatic magnetic stirrer was set to 65°C and the rotation speed to 300 rpm. -1 The silicon wafer with the pre-cut window is placed into the etching process and etched for 150 minutes to obtain the inverted pyramid pit silicon master mold.

[0017] Step S2: Prepare the pyramid microcone array SU-8 adhesive substrate by secondary molding and imprinting;

[0018] The preparation steps are as follows:

[0019] Take polymethyl methacrylate (PMMA) powder, add curing agent, and stir to dissolve until no obvious powder remains;

[0020] The silicon master mold obtained in step S1 is submerged at the bottom of the solution and then cured by heating.

[0021] After curing, the PMMA template is obtained by peeling.

[0022] Take polydimethylsiloxane (PDMS) prepolymer, add curing agent and stir, vacuum, and pour it onto PMMA template after there are no obvious bubbles;

[0023] After curing in an oven, the PDMS template is peeled off.

[0024] A layer of SU-8 photoresist is coated onto a PET film;

[0025] Pyramidal microcone array substrates were obtained by PDMS template imprinting.

[0026] The specific parameters in step S2 are as follows: Take 2g of polymethyl methacrylate (PMMA) powder, add 1.6g of curing agent, stir and dissolve until there is no obvious powder; sink the silicon master mold obtained in step S1 to the bottom of the solution, heat at 40° for 12h for curing, and peel off the PMMA template after curing; take 3g of polydimethylsiloxane (PDMS) prepolymer, add 0.3g of curing agent and stir, vacuum for 15min, pour it onto the PMMA template after there are no obvious bubbles, put it in a 70° oven for curing for two hours and peel off the PDMS template; coat a layer of SU-8 (3035) photoresist on a 150um PET film; use the PDMS template to imprint the pyramid microcone array substrate.

[0027] Step S3: Micro-nano fabrication to prepare rough nano-morphology on the surface of SU-8 adhesive substrate;

[0028] The SU-8 adhesive substrate obtained in step S2 was placed in a reactive ion etching machine for dry etching.

[0029] The etching power was adjusted to 350W, the O2 valve was opened, the air intake flow rate was adjusted to 20sccm, the etching pressure was adjusted to 5Pa, and the etching time was set to 15 minutes to obtain an SU-8 substrate with a nano-morphology on the surface.

[0030] Step S4: Electron beam evaporation of gold film;

[0031] In step S4, the electron beam evaporation of the gold film involves: attaching the prepared SU-8 adhesive substrate with its conical surface facing upwards onto the gold-plating disk, and controlling the gold evaporation process to... The rate was controlled by using a film thickness gauge to maintain the thickness of the evaporated gold layer on the substrate at approximately 50 nm.

[0032] Step S5: Modify the adaptor onto the SU-8 adhesive substrate;

[0033] In step S5, the step of modifying the aptamer on the SU-8 adhesive substrate is as follows: the SU-8 adhesive substrate is placed in a pre-prepared 70 nmol·L⁻¹ solution. -1 In the specific aptamer solution, in order to ensure that the aptamer fully contacts and modifies the substrate, the centrifuge tube is suspended at the edge of the vortex shaker. The tube is gently oscillated by colliding with the edge of the vortex shaker to avoid excessive oscillation. After oscillating for 24 hours, the tube is taken out and rinsed 2-3 times with PBS solution to obtain the SU-8 gel substrate incubated with the aptamer.

[0034] Step S6: Fabrication of the microfluidic chip.

[0035] In step S6, the microfluidic chip is prepared as follows: a positive template for the chip is prepared by photolithography; PDMS and curing agent are mixed in a ratio of 10:1 and stirred; vacuum is applied for 15 minutes; after no obvious bubbles are found, the mixture is poured into a petri dish containing the silicon positive template and placed in a 70-degree oven for two hours to cure; the PDMS chip is peeled off and cut into individual pieces to obtain the microfluidic chip.

[0036] Preferably, the present invention further includes step S7, sealing the embedded substrate chip, specifically: the microfluidic chip prepared in step S6 is ultrasonically cleaned with ethanol for 10 minutes, dried on a heating plate at 70 degrees, a glass slide is taken and placed together with the dried microfluidic chip into a Plasma plasma cleaner, vacuumed for 90 seconds, treated with oxygen plasma for 45 seconds, and the SU-8 adhesive substrate is embedded into the reserved cavity for sealing to obtain a complete microfluidic platform.

[0037] Preferably, the present invention further includes step S8, characterization of the SU-8 adhesive substrate. The characterization method is as follows: first, the size of individual pits in the inverted pyramid pit array silicon master mold is characterized using a white light interferometer. Then, the surface morphology of the SU-8 adhesive substrate is characterized using a scanning electron microscope. Finally, the SERS enhancement performance of the substrate is characterized using Raman reporter molecules. The substrate is immersed in malachite green isothiocyanate (MGITC) at different concentrations, and the results are detected using a Renishaw inVia microconfocal Raman spectroscopy system.

[0038] Advantages and beneficial effects of the present invention:

[0039] 1. The pyramidal microcone array SU-8 adhesive substrate has excellent SERS properties due to its micro-nano heterostructure. After modification of the aptamer, it combines the physical properties of the substrate with the affinity of the aptamer to achieve high CTCs capture efficiency.

[0040] 2. The combination of microfluidic chips and SERS substrates improves capture efficiency and provides a platform that can simultaneously capture and detect CTCs via SERS.

[0041] Terminology Explanation:

[0042] In this article, PS refers to polystyrene (PS) and PET refers to polyethylene terephthalate (PET).

[0043] SERS refers to the surface-enhanced Raman effect (SERS).

[0044] CTCs refer to circulating tumor cells.

[0045] PDMS refers to polydimethylsiloxane.

[0046] PMMA refers to polymethyl methacrylate, a high molecular weight polymer, also known as acrylic or plexiglass.

[0047] MGITC refers to malachite green isothiocyanate. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 A schematic diagram of the fabrication process of the silicon master mold for the pyramid pit array;

[0050] Figure 2 A schematic diagram of the fabrication process of the pyramidal microcone array SU-8 adhesive substrate;

[0051] Figure 3 Three-dimensional reconstruction image of a single inverted pyramidal recess in a silicon master mold and scanning electron microscope image of the SU-8 adhesive substrate;

[0052] Figure 4 Mapping diagrams of different concentrations of MGITC measured on SU-8 gel substrate;

[0053] Figure 5 Mapping diagram for uniformity detection of SERS substrate;

[0054] Figure 6 Fluorescence image of the pre-enrichment effect for deterministic lateral displacement;

[0055] Figure 7 A schematic diagram of CTCs captured within a microfluidic chip;

[0056] Figure 8 Fluorescence image of circulating tumor cells statically incubated on SU-8 gel substrate. Detailed Implementation

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

[0058] Step S1, fabrication of the inverted pyramid pit array silicon master mold;

[0059] Figure 1 This is a schematic diagram illustrating the fabrication process of the pyramid-shaped pit array silicon master mold in this invention. (See attached diagram.) Figure 1As shown, the specific method for preparing the inverted pyramid pit array silicon master mold in step S1 is as follows: photolithography is performed on a silicon wafer with a 300nm silicon dioxide (SiO2) oxide film to obtain square windows with a side length of 160um, a spacing of 30um, and staggered arrangement in each column. The exposed SiO2 film inside the window is removed using hydrofluoric acid, and the pattern is transferred to the SiO2 film layer.

[0060] Then, anisotropic etching is performed to obtain the inverted pyramidal pit silicon master mold. Specifically, 10.5 ml of a 6 mol·L⁻¹ potassium hydroxide (KOH) solution is taken, and 1 ml of isopropanol (IPA) and 1 ml of deionized water are added to obtain a 5 mol·L⁻¹ solution. -1 KOH anisotropic etching solution was used. The temperature of the constant temperature magnetic stirrer was set to 65°C and the speed was 300 rpm. The silicon wafer with the pre-cut window was placed in the stirring and etched for 150 minutes to obtain the inverted pyramid pit silicon master mold.

[0061] Step S2: Prepare the pyramid microcone array SU-8 adhesive substrate by secondary molding and imprinting;

[0062] Figure 2 This is a schematic diagram of the preparation process of the pyramid microcone array SU-8 adhesive substrate in this invention, as shown below. Figure 2 The specific method for preparing the pyramid microcone array SU-8 photoresist substrate by secondary molding and imprinting in step S2 is as follows: Take 2g of polymethyl methacrylate (PMMA) powder, add 1.6g of curing agent, and stir to dissolve until no obvious powder remains. Submerge the silicon master mold obtained in step S1 into the bottom of the solution, heat at 40°C overnight for curing, and peel off the PMMA template after curing. Take 3g of polydimethylsiloxane (PDMS) prepolymer, add 0.3g of curing agent and stir, vacuum for 15min, and after no obvious bubbles remain, pour it onto the PMMA template, place it in a 70°C oven for curing for two hours, and peel off the PDMS template. Coat a layer of SU-8 (3035) photoresist on a 150um PET film. Imprint the pyramid microcone array substrate using the PDMS template.

[0063] Step S3: Micro-nano fabrication to prepare rough nano-morphology on the surface of SU-8 adhesive substrate;

[0064] The prepared SU-8 adhesive substrate was placed in a reactive ion etching (RIE) machine for RIE etching. The etching power was adjusted to 350W, the O2 valve was opened, the inlet gas flow rate was adjusted to 20 sccm, the etching pressure was adjusted to 5 Pa, and the etching time was set to 15 minutes to obtain an SU-8 adhesive substrate with a nano-morphology on the surface.

[0065] Figure 3Figures a and b show a 3D reconstructed image of a single inverted pyramidal recess in the silicon master mold and a scanning electron microscope (SEM) image of the SU-8 adhesive substrate. Figures a and b show that the recess base has a side length of 160 μm, a height of approximately 70 μm, and a sidewall tilt angle of approximately 55°. Figure c is a SEM image of a single pyramidal cone on the SU-8 adhesive substrate, and Figure d is a magnified view of the sidewall.

[0066] Step S4: Electron beam evaporation of gold film;

[0067] The prepared SU-8 adhesive substrate, cone-shaped and facing upwards, was attached to the gold-plated disk, and the gold evaporation was controlled at 0.03 nm·s. -1 The rate was controlled by using a film thickness gauge to maintain the thickness of the evaporated gold layer on the substrate at approximately 50 nm.

[0068] Step S5: Modify the adaptor onto the SU-8 adhesive substrate;

[0069] First, the SU-8 adhesive substrate is placed in a plasma cleaner to improve its hydrophilicity. The valve is closed, and a vacuum is applied for approximately 90 seconds. Then, the knob is turned to "HI" and oxygen plasma treatment is performed using ultraviolet light for approximately 45 seconds. Next, the gas is slowly released from the chamber to prevent the substrate from being blown away by excessively rapid gas inflow. Afterward, the substrate is removed and placed in a pre-prepared 70 nmol / L... -1 In the Wy5a aptamer solution, to ensure sufficient contact and modification between the aptamer and the substrate, the centrifuge tube was suspended at the edge of a vortex mixer. The tube was gently vibrated by colliding with the edge of the mixer, preventing excessive vibration. After vibrating for 2 hours, the tube was rinsed 2-3 times with PBS solution. Finally, it was stored in a humid environment to obtain the SERS microcone SU-8 gel substrate with the aptamer incubated.

[0070] Step S6, fabrication of the microfluidic chip;

[0071] First, a microfluidic chip anode template is prepared using photolithography and fixed to the bottom of a culture dish using adhesive tape. A specially machined 230µm thick stainless steel gasket is placed on the capture chamber of the anode template surface. This gasket is removed when the PDMS chip is peeled off. The gasket increases the height of the chip chamber, allowing the SU-8 adhesive substrate to embed well. A PDMS mixture is prepared by mixing polydimethylsiloxane (PDMS) prepolymer and curing agent at a ratio of 10:1 and poured into the culture dish with the anode template fixed. The dish is placed on a heating plate and heated to 70°C for approximately 10 minutes. The position of the gasket is adjusted to ensure it fits tightly against the chamber area. During heating, some air bubbles may appear; these are promptly blown away with a bulb syringe to ensure the culture dish is free of air bubbles. After removing the air bubbles, the culture dish is placed flat in an oven at 70°C and baked for approximately two hours to completely solidify the PDMS mixture, resulting in a transparent, flexible solid. The cured PDMS was peeled off with a scalpel, and then the chip was cut along its edges with a utility knife. A hole was punched straight down at the designated location on the chip for subsequent solution entry and exit. This completed the fabrication of the microfluidic chip.

[0072] Step S7, Encapsulation of the embedded substrate chip;

[0073] The prepared microfluidic chip was ultrasonically cleaned with ethanol for 10 minutes. After drying, it was placed in a plasma cleaner PDC-32G-2. The air inside the plasma cleaner chamber was first evacuated for about 90 seconds. Then, the knob was turned to "HI" and oxygen plasma treatment was performed with ultraviolet light for 45 seconds. After that, the modified SU-8 adhesive substrate was embedded into the reserved capture chamber and then sealed to complete the chip assembly.

[0074] Step S8, characterization of the SU-8 adhesive substrate;

[0075] First, the size of individual pits in the inverted pyramid pit array silicon master mold was characterized using a white light interferometer. Then, the surface morphology of the SU-8 adhesive substrate was characterized using a scanning electron microscope. Finally, the SERS enhancement performance of the substrate was characterized using Raman reporter molecules. The substrate was immersed in different concentrations of malachite green isothiocyanate (MGITC), and the results were detected using a Renishaw inVia microconfocal Raman spectroscopy system.

[0076] Figure 4 This is a mapping diagram of different concentrations of MGITC measured on SU-8 adhesive substrates. First, five groups of gold-plated substrates were hydrophilically treated, and then immersed in 10... -6 ~10 -10The sample was immersed in MGITC solution for two hours. After removal, the sample was rinsed with deionized water to remove free reporter molecules. Mapping measurements were then performed using the LiveTrack function of the Renishaw inVia microconfocal Raman spectrometer. This function provides real-time focusing and is well-suited for measuring samples where the focal points are not on the same plane. The test conditions were: integration time 1 s, integration count 1, laser intensity 10%, total mapping measurement area 70 μm * 30 μm, and step size 2.5 μm. (From left to right, the values ​​are 10...) -6 ~10 -10 Mapping image obtained at the specified concentration.

[0077] Figure 5 This is a mapping image for the homogeneity detection of the SERS substrate, obtained by immersing the substrate in a solution of 10... -6 mol〃L -1 The sample was immersed in MGITC solution for 2 hours, then rinsed with deionized water and air-dried. The test conditions were: characterization area size 100µm * 100µm, step size 5µm, integration time 1s, laser intensity 10%, and one integration attempt. Figure 5 It can be seen that the entire single cone has a good SERS enhancement effect.

[0078] Figure 6 This is a fluorescence image of the pre-enrichment effect for deterministic lateral displacement, using PS fluorescent microspheres to simulate CTCs and erythrocytes in human blood. 15µm green fluorescent PS microspheres simulate cancer cells, and 6µm red fluorescent PS microspheres simulate erythrocytes. The fluorescence intensity is 10µl / min. -1 ~40ul〃min -1 The flow rate is introduced into the chip. Figure a shows a flow rate of 10 μL / min. -1 Figure b is 20ul〃min -1 Figure c is 30ul〃min -1 Figure d is 40ul〃min -1 As the flow rate increases, up to 30 μL / min... -1 The aggregation effect begins to disappear at that time. And 10ul〃min -1 20ul〃min -1 They all have excellent aggregation effects.

[0079] Figure 7This is a schematic diagram of CTCs capture within a microfluidic chip. The designed chip mainly consists of two inlet ports (a and b), three outlet ports (c and d), a pre-enrichment chamber (i), a separation region (ii), and a capture chamber (iii). First, approximately 100 CTCs (5 μL) are added to 95 μL of PBS buffer. This buffer is then introduced into the chip through inlet port (a) at a flow rate of 10 μL / min. After passing through the pre-enrichment chamber (i), the CTCs enter the capture chamber (iii) through the central channel of the separation region, thus achieving pre-enrichment of the CTCs. This splitting method also reduces the flow rate of the CTCs reaching the capture chamber, thereby improving the capture efficiency. After 100 μL of PBS buffer containing CTCs has been introduced, the chip is allowed to stand for half an hour. Then, PBS buffer is introduced through inlet port (b) at a flow rate of 10 μL / min to flush away any CTCs that failed to adhere to the substrate. Finally, a detection probe is introduced through port (b) at a flow rate of 1 μL / min. After the detection probe has passed through, the free detection probe is flushed away with PBS buffer, and finally, CTCs are analyzed using SERS.

[0080] Figure 8 This is a fluorescence image of circulating tumor cells statically incubated on a SU-8 gel substrate. First, a concentration of 10... 5 ~10 6 Centrifuge 1 ml of cell suspension (cells / mL) at 1000 rpm for 5 min, and collect 950 μL of supernatant. Add 950 μL of PBS buffer. Then, take 25 μL of cell suspension and add 1 μL of a live / dead cell staining solution. Let stand for 5 min to complete staining. Take 10 μL of the stained cell suspension and add it to a SU-8 gel substrate, incubate in the dark for 2 hours. The results are as follows. Figure 8 As shown.

[0081] All equivalent variations and modifications made to the spectral data contained herein according to the claims of this invention are covered by the scope of the claims of this invention.

Claims

1. A microfluidic chip embedded in an SU-8 gel substrate for CTCs capture and SERS analysis, characterized in that, The fabrication of the microfluidic chip includes the following steps: Step S1, preparation of the inverted pyramid pit array silicon mother film; Step S10: Photolithography is performed on the silicon wafer with silicon dioxide SiO2 oxide film to obtain square windows with a side length of 160um, a spacing of 30um and staggered arrangement in each column; Step S11: Use hydrofluoric acid to remove the SiO2 film exposed inside the window; In step S12, the pattern is transferred to the SiO2 film layer; Step S13: Add isopropanol (IPA) and deionized water to potassium hydroxide (KOH) solution to obtain KOH anisotropic etching solution; Step S14: Place the silicon wafer with the pre-cut window inside the magnetic stirrer and etch it to obtain the inverted pyramid pit silicon master mold. Step S2: Prepare the pyramid microcone array SU-8 adhesive substrate by secondary molding and imprinting; The preparation steps are as follows: Take polymethyl methacrylate (PMMA) powder, add curing agent, and stir to dissolve until no obvious powder remains; The silicon master mold obtained in step S1 is submerged at the bottom of the solution and then cured by heating. After curing, the PMMA template is obtained by peeling. Take polydimethylsiloxane PDMS prepolymer, add curing agent and stir, vacuum, and pour it onto PMMA template after there are no obvious bubbles. After curing in an oven, the PDMS template is peeled off. A layer of SU-8 photoresist is coated onto a PET film; Pyramidal microcone array substrates were obtained by PDMS template imprinting. Step S3: Micro-nano fabrication to prepare rough nano-morphology on the surface of SU-8 adhesive substrate; The SU-8 adhesive substrate obtained in step S2 was placed in a reactive ion etching machine for dry etching. Step S4: Electron beam evaporation of gold film; Step S5: Modify the fitter onto the gold-plated SU-8 adhesive substrate; Step S6, fabrication of the microfluidic chip; The microfluidic chip is prepared by photolithography to create a positive template for the chip. PDMS and curing agent are mixed in a 10:1 ratio and stirred. After vacuuming for 15 minutes and removing any obvious bubbles, the mixture is poured into a petri dish containing the silicon positive template and baked in a 70-degree oven for two hours to cure. The PDMS chip is then peeled off and cut into individual pieces to obtain the microfluidic chip. Step S7, sealing the embedded substrate chip, specifically: the microfluidic chip prepared in step S6 is ultrasonically cleaned with ethanol for 10 min, dried on a heating plate at 70 degrees Celsius, a glass slide is taken and placed together with the dried microfluidic chip into a Plasma plasma cleaner, vacuumed for 90 s, treated with oxygen plasma for 45 s, and the SU-8 adhesive substrate is embedded into the reserved cavity for sealing to obtain a complete microfluidic platform.

2. The microfluidic chip for CTCs capture and SERS analysis embedded in an SU-8 adhesive substrate as described in claim 1, characterized in that, The specific parameters in step S1 are as follows: photolithography is performed on a silicon wafer with a 300 nm thick silicon dioxide (SiO2) oxide film to obtain square windows with a side length of 160 μm, a spacing of 30 μm, and staggered arrangement in each column; the exposed SiO2 film inside the window is removed using hydrofluoric acid, and the pattern is transferred to the SiO2 film layer; 6 mol·L -1 10.5 ml of potassium hydroxide (KOH) solution was mixed with 1 ml of isopropanol (IPA) and 1 ml of deionized water to obtain a 5 mol·L⁻¹ solution. -1 KOH anisotropic etching solution; the temperature of the constant-temperature magnetic stirrer was set to 65°C and the rotation speed to 300 rpm·min. -1 The silicon wafer with the pre-cut window is placed into the etching process and etched for 150 minutes to obtain the inverted pyramid pit silicon master mold.

3. The microfluidic chip for CTCs capture and SERS analysis embedded in an SU-8 adhesive substrate as described in claim 1, characterized in that, The specific parameters in step S2 are as follows: Take 2g of polymethyl methacrylate (PMMA) powder, add 1.6g of curing agent, stir and dissolve until there is no obvious powder; immerse the silicon master mold obtained in step S1 into the bottom of the solution, heat at 40°C for 12h for curing, and peel off to obtain the PMMA template after curing; take 3g of polydimethylsiloxane (PDMS) prepolymer, add 0.3g of curing agent and stir, vacuum for 15min, pour it onto the PMMA template after there are no obvious bubbles, place it in a 70°C oven for two hours to cure, and peel off to obtain the PDMS template; coat a layer of 3035 SU-8 photoresist on a 150um PET film; use the PDMS template to imprint the pyramid microcone array substrate.

4. The microfluidic chip for CTCs capture and SERS analysis embedded in an SU-8 adhesive substrate as described in claim 1, characterized in that, Specifically, step S3 involves adjusting the etching power to 350 W, opening the O2 valve, adjusting the inlet air flow rate to 20 sccm, adjusting the etching pressure to 5 Pa, and setting the etching time to 15 minutes to obtain an SU-8 substrate with a nano-morphology on the surface.

5. The microfluidic chip for CTCs capture and SERS analysis embedded in an SU-8 adhesive substrate as described in claim 1, characterized in that, In step S4, the electron beam evaporation of the gold film is performed as follows: the prepared SU-8 adhesive substrate is attached to the gold-plated disk with the conical surface facing upward, the gold evaporation rate is controlled at 0.5 Å, and the thickness of the gold layer on the substrate is controlled to be 50 nm using a film thickness gauge.

6. The microfluidic chip for CTCs capture and SERS analysis embedded in an SU-8 adhesive substrate as described in claim 1, characterized in that, In step S5, the step of modifying the aptamer on the SU-8 adhesive substrate is as follows: placing the SU-8 adhesive substrate into a pre-prepared 70 nmol·L⁻¹ solution. -1 In the specific aptamer solution, in order to ensure that the aptamer fully contacts and modifies the substrate, the centrifuge tube is suspended at the edge of the vortex shaker. The tube is gently oscillated by colliding with the edge of the vortex shaker to avoid excessive oscillation. After oscillating for 24 hours, the tube is taken out and rinsed 2-3 times with PBS solution to obtain the SU-8 gel substrate incubated with the aptamer.

7. The microfluidic chip for CTCs capture and SERS analysis embedded in an SU-8 adhesive substrate as described in claim 1, characterized in that, It also includes step S8, characterization of the SU-8 adhesive substrate, the characterization method being: First, the size of a single pit in the silicon master model of the inverted pyramid pit array is characterized by a white light interferometer. The surface morphology of the SU-8 adhesive substrate was then characterized using scanning electron microscopy. Finally, the SERS enhancement performance of the substrate was characterized by Raman reporter molecules. The substrate was immersed in different concentrations of malachite green isothiocyanate (MGITC), and the results were detected using a Renishaw inVia microconfocal Raman spectrometer.

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