Preparation method and application of anti-biological non-specific adsorption Raman enhanced substrate

Abstract

The invention discloses a preparation method and application of an anti-biological nonspecific adsorption Raman enhanced substrate. The preparation method comprises the following steps: 1) preparing a substrate seal by adopting an ultraviolet lithography and reverse molding method; step 2), preparing a DLD triangular prism array substrate; 3) processing the surface structure of the DLD triangular prism array substrate; (4) plating gold on the substrate of the DLD triangular prism array; step 5), performing SEM (scanning electron microscope) representation on a DLD triangular prism array substrate; 6) testing the SERS performance of the substrate; (7) incubating a substrate aptamer; (8) zwitterionic synthesis and modification; the zwitterions provided by the invention can reduce non-specific adsorption of substances on the substrate, so that CTCs can be in better contact with the substrate, and the substrate provided by the invention has more micro-nano structures, has a higher specific surface area, and can better promote cells to stretch out and adhere to pseudopods, so that the CTCs can be effectively adsorbed to the substrate. Meanwhile, the huge specific surface area can modify more aptamers to capture tumor cells, so that the capture efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the field of micro-nano sensing and microfluidics technology. Specifically, the present invention relates to a method for preparing a Raman-enhanced substrate resistant to biological nonspecific adsorption and its application. Background Art

[0002] Surface-enhanced Raman scattering technology is a non-destructive and highly sensitive detection method with the characteristics of fingerprints. Surface-enhanced Raman technology was first reported by American scholars Fleischmann et al. in 1974. He found that the Raman signal of pyridine adsorbed on the surface of a rough silver electrode was significantly enhanced, but he believed that this was because the increase in the surface area of ​​the rough electrode led to an increase in the number of adsorbed pyridine molecules, resulting in signal enhancement. Subsequently, Van Duyne et al. and Creighton et al. concluded after rigorous experiments and calculations that the signal enhancement of pyridine on a roughened silver electrode came from a certain enhancement effect rather than a simple increase in surface area. This effect was later called surface-enhanced Raman Scattering (SERS).

[0003] Femtosecond laser is a laser with extremely short pulse width, usually referring to a laser with pulse duration in the femtosecond range. Femtosecond laser has extremely high peak power and can release a large amount of energy in a very short time. Therefore, femtosecond laser can ablate any material. When femtosecond laser irradiates the surface of the material, the laser pulse photons are absorbed by the material to form hot electrons. The hot electrons collide with the material lattice to remove the material, thereby processing micro-nano structures on the surface of the material.

[0004] Inductively coupled plasma etching is a type of dry etching. The inductively coupled plasma etching system uses upper and lower electrodes that are independently controlled by two RF power supplies. The upper electrode controls the generation of plasma, and the lower electrode controls the kinetic energy of the plasma. The plasma reacts chemically with the material surface to form volatile gases, which can be extracted to complete the etching of the material. However, materials such as metals cannot be etched, thereby forming micro-nano structures on the material surface.

[0005] Zwitterions are substances that contain both positively and negatively charged groups in the unit and are neutrally charged overall. They exhibit strong hydration ability. Zwitterionic materials interact with water molecules through electrostatically induced hydration (or ion solvation) to form a solid hydration layer on the surface of the material and have been used in a wide range of biomedical applications, including biosensors, medical devices, cell encapsulation, drug delivery, protein modification, etc. Summary of the invention

[0006] The purpose of the present invention is to solve the problem of nonspecific adsorption of traditional SERS substrate materials and fluid flowing away from the gap between the embedded substrate and the chamber, and to provide a nonspecific adsorption resistant Raman enhancement substrate, as well as the preparation and application of the substrate in biological detection.

[0007] To achieve the above purpose, the technical solution provided by the present invention is:

[0008] A method for preparing a substrate for anti-biological nonspecific adsorption Raman enhancement comprises the following steps;

[0009] Step 1), preparing a base stamp by using ultraviolet lithography and a reverse molding method;

[0010] Draw the triangular prism base of deterministic lateral displacement (DLD) using AutoCAD and print out the film version;

[0011] Spin-coat 30 μm 3035 photoresist on the silicon wafer and prepare a positive template by photolithography;

[0012] The base stamp was obtained by polydimethylsiloxane (PDMS) casting;

[0013] Step 2), DLD triangular prism array substrate preparation;

[0014] Drop a drop of SU-2010 photoresist on a glass sheet, press the stamp prepared in step 1) on the photoresist, and photocuring to obtain a DLD triangular prism array substrate;

[0015] Step 3), DLD triangular prism array substrate surface structure processing;

[0016] The DLD triangular prism array substrate of step 2) is processed by femtosecond laser under optimized femtosecond laser parameters, and then the inductively coupled plasma is optimized to process a micro-nano structure on the side wall of the triangular prism to obtain a DLD triangular prism array substrate with a micro-nano structure;

[0017] Step 4), DLD triangular prism array substrate is gold plated;

[0018] The processed substrate was gold-coated to 30 nm in an electron beam vacuum coating apparatus;

[0019] Step 5), SEM characterization of DLD triangular prism array substrate;

[0020] The surface morphology of the triangular micropillar substrates processed under different conditions was characterized by thermal field emission scanning electron microscopy observation and recording of the surface morphology;

[0021] Step 6), SERS performance test of the substrate;

[0022] The gold-plated DLD triangular prism array substrate of step 4) was subjected to oxygen plasma hydrophilization treatment, and then placed in MGITC solution, and the uniformity of the substrate SERS performance was tested using a confocal Raman spectrometer;

[0023] Step 7), substrate aptamer incubation;

[0024] Step 8), zwitterion synthesis and modification;

[0025] Zwitterionic N,N-bis(3-aminopropyl)methylamine oxide(DNMAO) was synthesized by oxidizing N,N-bis(3-aminopropyl)methylamine(DNMA) and modified on the substrate.

[0026] The preferred technical solution provided by the present invention is: in the step 1), specifically:

[0027] First, 30μm SU-3035 photoresist was spin-coated on the cleaned silicon wafer, and exposed through a mask in a UV photolithography machine for 40s. Three exposures were used, each for about 13s, and then the pattern was post-baked at 95℃ for 2min to reveal the pattern. The positive template of the substrate was obtained by development. PDMS and curing agent were mixed in a ratio of 10:1, stirred evenly, and bubbles were removed in a vacuum machine. The prepared positive template was injection molded, cured in a 70℃ oven for 1-2h, and cut with a cutter to obtain a PDMS stamp.

[0028] The triangular micro-pillars prepared from the triangular prism substrate of the deterministic lateral displacement (DLD) are 30 μm high, 40 μm long, with an offset angle of 3-5° and a critical diameter of 15 μm.

[0029] The preferred technical solution provided by the present invention is: the step 2) is specifically:

[0030] Add a drop of SU-2010 photoresist on the glass slide, and imprint the stamp made in step 1) on the photoresist. After standing for about 5 minutes, turn it over and stand for about 5 minutes again to make the photoresist full of the stamp pattern. Finally, expose it in a UV box for 60 to 120 seconds.

[0031] The further preferred technical solution provided by the present invention is:

[0032] The specific method of processing the DLD triangular array substrate surface structure in step 3) is as follows:

[0033] Processing the substrate processed in step 2) with a femtosecond laser at 18-32 mW, 5 kHz frequency, and 5 mm / s scanning speed to ablate the surface of the material;

[0034] Then, etching was performed in an inductively coupled plasma etcher, wherein the etching parameters were upper radio frequency 150 W, lower radio frequency 50 W, oxygen flow rate set to 30 sccm, helium 20 sccm, and etching time 7.5 min;

[0035] Thereby, micro-nano structures are etched on the sidewalls.

[0036] Another preferred technical solution provided by the present invention is:

[0037] The step 6) is specifically as follows:

[0038] Soak the substrate in 10 -6 mol / L MGITC solution for 10-12 hours, take out and air-dry for testing;

[0039] The Raman spectrometer used was a confocal Raman spectrometer Renishaw Invia, and the parameters of the spectrometer were set as follows: the excitation light source was 633 nm, the microscope objective lens was 50× (0.5 NA), the laser power was 2.5 to 5 mW, the integration time was 1 s, and the number of integrations was 1.

[0040] A further preferred technical solution provided by the present invention is:

[0041] The step 7) is specifically as follows:

[0042] The gold-plated substrate prepared in step 4) is placed in a plasma cleaning machine for 2 minutes to make the substrate have good hydrophilicity;

[0043] The treated substrate was immersed in a pre-prepared 70 nmol / L specific aptamer solution and then hung on the edge of a honeycomb oscillator for 20 to 24 hours.

[0044] Finally, the substrate soaked with the aptamer was rinsed 2 to 3 times with PBS solution to remove the free aptamer on the surface.

[0045] The more preferred technical solution provided by the present invention is:

[0046] The step 8) is specifically as follows:

[0047] Add 15 ml of 8% hydrogen peroxide solution dropwise to 20 ml of 34.5 mmol / L DNMA aqueous solution in an ice-water bath, stir the final solution in an ice-water bath for 10 to 12 hours, extract the reaction solution with dichloromethane 2 to 3 times to obtain DNMAO;

[0048] The substrate modified with the aptamer in step 7) was soaked in a 1 mmol / L 11-mercaptoundecanoic acid solution (11-MUA) for 1-2 h, and then the substrate was taken out and the carboxyl group was activated in a 1 mmol / L mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Then, the synthesized DNMAO was added dropwise to the substrate to react for 1-2 h, and finally, the substrate was washed with a PBS solution 1-2 times to remove unbound molecules.

[0049] The present invention also provides a technical solution for applying a substrate prepared by a method for preparing a Raman-enhanced substrate resistant to biological nonspecific adsorption in a microfluidic chip.

[0050] The present invention also provides a technical solution for the application of a substrate prepared by a method for preparing a Raman-enhanced substrate resistant to biological nonspecific adsorption in the detection of circulating tumor cells.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. Anti-biological non-specific adsorption Raman enhanced substrate can reduce the non-specific adsorption of biological substances such as blood cells and proteins, and improve the binding efficiency between cells and substrates.

[0053] 2. The substrate has a high Raman signal enhancement effect.

[0054] Terminology explanation:

[0055] DLD——Deterministic Lateral Displacement;

[0056] PDMS——Polydimethylsiloxane;

[0057] MGITC——Malachite Green Isothiocyanate;

[0058] PBS solution——Phosphate Buffer Solution;

[0059] DNMA——N,N-bis(3-aminopropyl)methylamine;

[0060] DNMAO——N,N-bis(3-aminopropyl)methylamine N-oxide;

[0061] EDC——1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0062] (3-dimethylaminopropyl)carbodiimide);

[0063] NHS——N-Hydroxysuccinimide; BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The present invention provides a preparation process of the triangular prism array substrate;

[0065] Figure 2 SEM images of the substrate after imprint curing, femtosecond laser ablation, and ICP etching;

[0066] Figure 3 The SERS spectra of MGITC measured at different etching times for the triangular prism array SERS substrate;

[0067] Figure 4 This is the uniformity and reproducibility test diagram of the triangular prism array SERS substrate;

[0068] Figure 5 These are the UV absorption spectra of the gold-plated substrate, modified aptamer, and modified zwitterion substrate. DETAILED DESCRIPTION

[0069] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings.

[0070] Figure 1 1 is a flow chart of the preparation of the triangular prism array substrate of the present invention. Figure 1 The specific instructions are as follows:

[0071] First, a triangular column array is obtained by photolithography, and the micro-nano structure on the surface of the micro-column is increased by femtosecond laser and inductively coupled plasma etching, thereby increasing the specific surface area of ​​the substrate to facilitate subsequent cell attachment. A gold film is electron beam evaporated on the substrate, so that the substrate can have more hotspots and have a better Raman signal enhancement effect.

[0072] The specific steps of the present invention are:

[0073] Step 1), preparation of a base stamp;

[0074] The substrate stamp was prepared by UV lithography and reverse molding. First, 30 μm SU-3035 photoresist was spin-coated on the cleaned silicon wafer, and the mask was exposed for 40 seconds in a UV lithography machine. Three exposures were used, each for about 13 seconds, and then the pattern was post-baked at 95°C for about 2 minutes to reveal the pattern. The positive template of the substrate was obtained by development. PDMS and curing agent were mixed in a ratio of 10:1, stirred evenly, and bubbles were removed in a vacuum machine. The prepared positive template was injection molded and reversed, and cured in a 70°C oven for 2 hours. The PDMS stamp was obtained by cutting with a cutter.

[0075] Step 2), DLD triangular prism array substrate preparation;

[0076] A drop of SU-2010 photoresist is dripped on the glass sheet, and the stamp made in step 1) is pressed on the photoresist. After standing for 5 minutes, it is turned over and then stood for another 5 minutes to make the photoresist full of the stamp pattern. Finally, it is exposed in a UV box for 60 seconds. SU-2010 photoresist is used because it is relatively thin and can fill the stamp pattern.

[0077] Step 3), DLD triangle array substrate surface structure processing;

[0078] In order to obtain a micro-nano structure on the side of the triangular column, the substrate processed in step 2) is processed by a femtosecond laser of 18 to 32 mW, and the laser scanning speed is 5 mm / s to ablate the surface of the material. In order to obtain a micro-nano structure on the side wall of the microcolumn, an inductively coupled plasma etcher with an upper radio frequency of 150 W and a lower radio frequency of 50 W is used for etching at the same time. The oxygen plasma in the etcher can react with the material to generate gas, thereby etching a micro-nano structure on the side wall.

[0079] In the step 3), in order to optimize the surface and sidewall micro-nanostructures, the femtosecond laser energy is optimized to 18mW, 24mW, 28mW, and 32mW, and the etching time is optimized to 150s, 300s, 450s, 600s, and 750s.

[0080] Figure 2 These are SEM images of the substrate after imprint curing, femtosecond laser ablation, and ICP etching. Figure 2 It can be seen that after laser ablation and ICP etching, the side of the triangular prism has a better micro-nano structure, which is more conducive to improving the adsorption capacity of the substrate.

[0081] Step 4), gold plating of the triangular prism array substrate;

[0082] In order to enable the substrate to enhance the Raman signal and facilitate the modification of the aptamer, a 30 nm gold film is evaporated on the substrate processed in step 3) by an electron beam vacuum coating apparatus.

[0083] Step 5), SEM characterization of the triangular prism base;

[0084] In order to characterize the differences in surface morphology of substrates under femtosecond laser and plasma etching times, substrates with different processing conditions were attached to the sample stage, metal was sprayed to increase the conductivity of the substrate, and the surface morphology was recorded by a scanning high-resolution scanning electron microscope.

[0085] Step 6), SERS performance test of the triangular prism substrate;

[0086] Figure 3 The SERS spectra of MGITC measured at different etching times for the triangular prism array SERS substrate; Figure 4 This is the uniformity and reproducibility test diagram of the triangular prism array SERS substrate; combined with Figure 3 , Figure 4 ,

[0087] The Raman signals of the substrates with different etching times were characterized to determine the optimal etching time for the substrate. The SERS substrates with different etching times were treated with oxygen plasma for about 2 minutes and immersed in 10 -6 mol / LMGITC solution for about 12 h, and the Raman measurement of the substrate was carried out under a microscopic confocal Raman spectrometer. The uniformity and reproducibility of the Raman signal are important indicators for measuring the SERS performance.

[0088] The soaked substrate was taken out and air-dried. Ten Raman spectra were measured under a Raman spectrometer at 2.5-5 mW, integration time of 1 s, and integration times of 1. At the same time, the characteristic peak of MGITC at 1614 cm was taken. -1 The relative standard deviation of the Raman intensity at is calculated to be 6.71%, indicating that the substrate has good uniformity.

[0089] Step 7), substrate aptamer incubation;

[0090] The substrate modified with aptamers is the basis for the substrate to capture CTCs. The gold-plated substrate under the best conditions was treated in a plasma cleaner for about two minutes, and then immersed in a 70nmol / L wy5a aptamer solution for 20 to 24 hours. The substrate and the aptamer were bound through the Au-S bond. In order to better bind the substrate and the aptamer, a honeycomb oscillator was used during the immersion process, and the free aptamer was removed by rinsing with PBS solution 2-3 times.

[0091] The method comprises step 8) zwitterion synthesis and modification;

[0092] Zwitterions N,N-bis(3-aminopropyl)methylamine oxide (DNMAO) was synthesized by oxidizing N,N-bis(3-aminopropyl)methylamine (DNMA) with hydrogen peroxide and then modified on the substrate.

[0093] The specific implementation steps are as follows: 15 ml of 8% hydrogen peroxide solution is added dropwise to 20 ml of 34.5 mmol / L DNMA aqueous solution in an ice-water bath, the final solution is stirred in an ice-water bath for 10 to 12 hours, the reaction-completed solution is extracted with dichloromethane 2 to 3 times to obtain an aqueous solution of DNMAO, and the aqueous solution is freeze-dried to obtain a DNMAO solution. At the same time, the substrate modified with the aptamer in step 7) is placed in a 1 mmol / L 11-mercaptoundecanoic acid tetrahydrofuran solution (11-MUA) for about 2 hours, 11MUA binds to the substrate through the AU-S bond, and then the substrate is taken out and the carboxyl group is activated in a 1 mmol / L mixed solution of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then the synthesized DNMAO is added dropwise to the substrate for reaction for about 2 hours, and DNMAO is modified on the substrate through the condensation reaction of the amino group and the carboxyl group, and finally the substrate is washed with PBS 1 to 2 times to remove unbound molecules.

[0094] Figure 5 The UV absorption spectra of the substrate at different modification stages are as follows:

[0095] Take a gold-plated substrate, modify the aptamer, cut the substrate modified with the aptamer and zwitterion into a suitable size, put it into an ultraviolet spectrophotometer to measure its absorption spectrum in the wavelength range of 200-800nm, and reflect the modification process of the aptamer and zwitterion through the spectral changes at different stages, so as to judge whether the aptamer and zwitterion are modified on the substrate.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a Raman enhanced substrate for resisting non-specific adsorption of organisms, characterized in that: The steps include: Step 1), preparing a base stamp by using ultraviolet lithography and a reverse molding method; Draw the triangular prism base of deterministic lateral displacement (DLD) using AutoCAD and print out the film version; Spin-coat 30 μm 3035 photoresist on the silicon wafer and prepare a positive template by photolithography; The base stamp was obtained by polydimethylsiloxane (PDMS) casting; Step 2), DLD triangular prism array substrate preparation; Drop a drop of SU-2010 photoresist on a glass sheet, press the stamp prepared in step 1) on the photoresist, and photocuring to obtain a DLD triangular prism array substrate; Step 3), DLD triangular prism array substrate surface structure processing; The DLD triangular prism array substrate of step 2) is processed by femtosecond laser under optimized femtosecond laser parameters, and then the inductively coupled plasma is optimized to process a micro-nano structure on the side wall of the triangular prism to obtain a DLD triangular prism array substrate with a micro-nano structure; Step 4), DLD triangular prism array substrate is gold plated; The processed substrate was gold-coated to 30 nm in an electron beam vacuum coating apparatus; Step 5), SEM characterization of DLD triangular prism array substrate; The surface morphology of the triangular micropillar substrates processed under different conditions was characterized by thermal field emission scanning electron microscopy observation and recording of the surface morphology; Step 6), SERS performance test of the substrate; The gold-plated DLD triangular prism array substrate of step 4) was subjected to oxygen plasma hydrophilization treatment, and then placed in MGITC solution, and the uniformity of the substrate SERS performance was tested using a confocal Raman spectrometer; Step 7), substrate aptamer incubation; Step 8), zwitterion synthesis and modification; Zwitterionic N,N-bis(3-aminopropyl)methylamine oxide(DNMAO) was synthesized by oxidizing N,N-bis(3-aminopropyl)methylamine(DNMA) and modified on the substrate.

2. The method for preparing a Raman enhanced substrate for anti-biological nonspecific adsorption according to claim 1, characterized in that: In the step 1), specifically: First, 30μm SU-3035 photoresist was spin-coated on the cleaned silicon wafer, and exposed through a mask in a UV photolithography machine for 40s. Three exposures were used, each for about 13s, and then the pattern was post-baked at 95℃ for 2min to reveal the pattern. The positive template of the substrate was obtained by development. PDMS and curing agent were mixed in a ratio of 10:1, stirred evenly, and bubbles were removed in a vacuum machine. The prepared positive template was injection molded, cured in a 70℃ oven for 1-2h, and cut with a cutter to obtain a PDMS stamp. The triangular micro-pillars prepared from the triangular prism substrate of the deterministic lateral displacement (DLD) are 30 μm high, 40 μm long, with an offset angle of 3-5° and a critical diameter of 15 μm.

3. The method for preparing a Raman enhanced substrate for anti-biological nonspecific adsorption according to claim 1, characterized in that: The step 2) is specifically as follows: Add a drop of SU-2010 photoresist on the glass slide, and imprint the stamp made in step 1) on the photoresist. After standing for about 5 minutes, turn it over and stand for about 5 minutes again to make the photoresist full of the stamp pattern. Finally, expose it in a UV box for 60 to 120 seconds.

4. The method for preparing a Raman enhanced substrate for resisting non-specific adsorption of organisms according to claim 1, characterized in that: The specific method of processing the DLD triangular array substrate surface structure in step 3) is as follows: Processing the substrate processed in step 2) with a femtosecond laser at 18-32 mW, 5 kHz frequency, and 5 mm / s scanning speed to ablate the surface of the material; Then, etching was performed in an inductively coupled plasma etcher, wherein the etching parameters were upper radio frequency 150 W, lower radio frequency 50 W, oxygen flow rate set to 30 sccm, helium 20 sccm, and etching time 7.5 min; Thereby, micro-nano structures are etched on the sidewalls.

5. The method for preparing a Raman enhanced substrate for resisting non-specific adsorption of organisms according to claim 1, characterized in that: The step 6) is specifically as follows: Soak the substrate in 10 -6 mol / L MGITC solution for 10-12 hours, take out and air-dry for testing; The Raman spectrometer used was a confocal Raman spectrometer Renishaw Invia, and the parameters of the spectrometer were set as follows: the excitation light source was 633 nm, the microscope objective lens was 50× (0.5 NA), the laser power was 2.5 to 5 mW, the integration time was 1 s, and the number of integrations was 1.

6. The method for preparing a Raman enhanced substrate resistant to biological nonspecific adsorption according to claim 1, characterized in that: The step 7) is specifically as follows: The gold-plated substrate prepared in step 4) is placed in a plasma cleaning machine for 2 minutes to make the substrate have good hydrophilicity; The treated substrate was immersed in a pre-prepared 70 nmol / L specific aptamer solution and then hung on the edge of a honeycomb oscillator for 20 to 24 hours. Finally, the substrate soaked with the aptamer was rinsed 2 to 3 times with PBS solution to remove the free aptamer on the surface.

7. The method for preparing a Raman enhanced substrate resistant to biological nonspecific adsorption according to claim 1, characterized in that: The step 8) is specifically as follows: Add 15 ml of 8% hydrogen peroxide solution dropwise to 20 ml of 34.5 mmol / L DNMA aqueous solution in an ice-water bath, stir the final solution in an ice-water bath for 10 to 12 hours, extract the reaction solution with dichloromethane 2 to 3 times to obtain DNMAO; The substrate modified with the aptamer in step 7) was soaked in a 1 mmol / L 11-mercaptoundecanoic acid solution (11-MUA) for 1-2 h, and then the substrate was taken out and the carboxyl group was activated in a 1 mmol / L mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Then, the synthesized DNMAO was added dropwise to the substrate to react for 1-2 h, and finally, the substrate was washed with a PBS solution 1-2 times to remove unbound molecules.

8. Use of a substrate prepared by the method for preparing a Raman-enhanced substrate resistant to non-specific biological adsorption according to any one of claims 1 to 7 in a microfluidic chip.

9. Use of a substrate prepared by the method for preparing a Raman-enhanced substrate resistant to non-specific biological adsorption according to any one of claims 1 to 7 in the detection of circulating tumor cells.

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