Preparation method of anti-bio non-specific adsorption raman enhancement substrate and application thereof

Abstract

The application discloses a kind of anti-biological nonspecific adsorption raman enhancement substrate preparation method and its application, including step 1), substrate stamp is prepared using ultraviolet lithography and reverse mold method;Step 2), DLD triangular prism array substrate preparation;Step 3), DLD triangular prism array substrate surface structure processing;Step 4), DLD triangular prism array substrate gold plating;Step 5), DLD triangular prism array substrate SEM characterization;Step 6), substrate SERS performance test;Step 7), substrate aptamer incubation;Step 8), zwitterion synthesis and modification;The zwitterion proposed in the application can reduce the nonspecific adsorption of substances on the substrate, so that CTCs can better contact the substrate, and the substrate of the application has more micro-nano structures, has higher specific surface area, better promotes cell pseudopod to adhere, and the huge specific surface area can modify more aptamers to capture tumor cells, thereby improving the capture efficiency.

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 that is resistant to biological nonspecific adsorption and its application. Background Art

[0002] Surface-enhanced Raman scattering (SERS) is a non-destructive, highly sensitive detection method with fingerprint-like properties. It was first reported in 1974 by American researchers Fleischmann et al., who found a significant enhancement in the Raman signal of pyridine adsorbed on a roughened silver electrode. However, they attributed this to an increase in the number of adsorbed pyridine molecules due to the increased surface area of ​​the roughened electrode. Subsequently, after rigorous experiments and calculations, Van Duyne et al. and Creighton et al. concluded that the enhanced signal of pyridine on a roughened silver electrode stems from a specific enhancement effect rather than simply an increase in surface area. This effect has since become known as surface-enhanced Raman scattering (SERS).

[0003] A femtosecond laser is a laser with an extremely short pulse width, typically in the femtosecond range. With its extremely high peak power, a femtosecond laser can release a large amount of energy in a very short time. Therefore, it can ablate any material. When a femtosecond laser strikes a surface, the laser pulse photons are absorbed by the material, forming hot electrons. These hot electrons collide with the material's crystal lattice, removing material and creating micro-nanostructures on the surface.

[0004] Inductively coupled plasma etching is a type of dry etching. The inductively coupled plasma etching system uses upper and lower electrodes independently controlled by two radio frequency 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 their units and are neutrally charged overall. They exhibit strong hydration ability. Zwitterionic materials interact with water molecules through electrostatically induced hydration (or ion solvation), forming a solid hydration layer on the surface of the material. They 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 application aims at solving the problems of non-specific adsorption of traditional SERS substrate and fluid flowing away from the embedded substrate and the gap of the chamber, and provides a non-specific adsorption-resistant Raman enhancement substrate, a preparation method of the substrate and application of the substrate in biological detection.

[0007] To achieve the above-mentioned purpose, the technical scheme provided by the application is:

[0008] A non-specific adsorption-resistant Raman enhancement substrate preparation method comprises the following steps:

[0009] Step 1), a substrate stamp is prepared by using ultraviolet lithography and reverse mold method;

[0010] A triangular column substrate of deterministic lateral displacement (DLD) is drawn by AutoCAD, and a film plate is printed out;

[0011] A positive template is prepared on a silicon wafer by spin-coating 30 mu m of 3035 photoresist and photoetching;

[0012] The substrate stamp is obtained by polydimethylsiloxane (PDMS) reverse mold;

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

[0014] A drop of SU-2010 photoresist is dropped on a glass sheet, the stamp prepared in step 1) is pressed on the photoresist, and a DLD triangular column array substrate is obtained by light curing;

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

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

[0017] Step 4), DLD triangular column array substrate gold plating;

[0018] The processed substrate is gold-plated for 30 nm in an electron beam vacuum coating instrument;

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

[0020] The surface morphology of the triangular micro-column substrate processed under different processing conditions is characterized, and the surface morphology is observed by thermal field emission scanning electron microscopy and recorded;

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

[0022] After the gold-plated DLD triangular prism array substrate of step 4) is subjected to oxygen plasma hydrophilic treatment, it is placed into the MGITC solution, and the substrate SERS performance uniformity test is carried out by using a confocal Raman spectrometer;

[0023] Step 7), substrate aptamer incubation;

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

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

[0026] The preferred technical scheme provided by the application is that:

[0027] First, 30 mu m of SU-3035 photoresist is spin-coated on the cleaned silicon wafer, exposed to UV ultraviolet light for 40s through a mask, and three times of exposure is adopted, about 13s each time, then post-baking at 95 DEG C for 2min to make the pattern appear, and the positive template of the substrate is obtained by developing; the PDMS and curing agent 10:1 are mixed, stirred uniformly, and the bubbles are removed in a vacuum machine, the prepared positive template is injection molded, and the PDMS stamp is obtained by cutting in a 70 DEG C oven for 1-2h.

[0028] The triangular microcolumn with a height of 30 mu m, a triangular prism side length of 40 mu m, an offset angle of 3-5 DEG, and a critical diameter of 15 mu m is prepared by the triangular prism substrate of the deterministic lateral displacement (DLD).

[0029] The preferred technical scheme provided by the application is that:

[0030] A drop of SU-2010 photoresist is added on the glass sheet, the stamp prepared in step 1) is pressed on the photoresist, and after standing for about 5min, it is turned over and stood for about 5min, so that the photoresist fills the stamp pattern, and finally exposed to UV for 60-120s.

[0031] The further preferred technical scheme provided by the application is:

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

[0033] The substrate processed in step 2) is processed by a femtosecond laser with a power of 18-32mW, a frequency of 5k Hz, and a scanning speed of 5mm / s, and the material surface is ablated;

[0034] Then etching is carried out in an inductively coupled plasma etching machine, wherein the etching parameters are: upper radio frequency 150W, lower radio frequency 50W, oxygen flow rate 30sccm, helium flow rate 20sccm, etching time 7.5min;

[0035] Thus, the micro-nano structure is etched on the side wall.

[0036] The application further provides another preferred technical scheme, which is as follows:

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

[0038] The substrate is soaked in 10 -6 mol / L MGITC solution for 10-12h, and then taken out for air drying and testing.

[0039] The Raman spectrometer is a confocal Raman spectrometer Renishaw Invia, and the parameter settings of the spectrometer are as follows: excitation light source is 633nm, 50x microscope objective (0.5NA), laser power is 2.5-5mW, integration time is 1s, and integration number is 1.

[0040] The application further provides a further preferred technical scheme, which is as follows:

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

[0042] The gold-plated substrate prepared in the step 4) is put into a plasma cleaning machine for treatment for 2min, so that the substrate has good hydrophilicity.

[0043] The specific aptamer solution with a concentration of 70nmol. / L is prepared in advance, then the treated substrate is soaked in the aptamer solution, and is hung on the edge of a honeycomb oscillator for oscillation for 20-24h.

[0044] Finally, the substrate after soaking the aptamer is washed with PBS solution for 2-3 times, so as to remove the free aptamer on the surface.

[0045] The application further provides a more preferred technical scheme, which is as follows:

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

[0047] 15ml 8% hydrogen peroxide solution is added dropwise in 20ml DNMA aqueous solution with a concentration of 34.5mmol / L under ice water bath, the last solution is stirred under ice water bath for 10-12h, the reaction complete solution is extracted with dichloromethane for 2-3 times, and DNMAO is obtained.

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

[0049] The application also provides a technical scheme of application of the substrate prepared by the preparation method of the anti-bio non-specific adsorption Raman enhancement substrate in a microfluidic chip.

[0050] The application also provides a technical scheme of application of the substrate prepared by the preparation method of the anti-bio non-specific adsorption Raman enhancement substrate in detection of circulating tumor cells.

[0051] Compared with the prior art, the application has the beneficial effects that:

[0052] 1. The anti-bio non-specific adsorption Raman enhancement substrate can reduce non-specific adsorption of biological substances such as blood cells and proteins, and improve the binding efficiency of cells and the substrate.

[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 (1-Ethyl-3-

[0062] (3-dimethylaminopropyl)carbodiimide);

[0063] NHS - N-hydroxysuccinimide (N-Hydroxysuccinimide); BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 is the preparation process of the triangular column array substrate of the present application;

[0065] Figure 2 is the SEM image of the substrate after imprinting and curing, after femtosecond laser ablation, and after ICP etching;

[0066] Figure 3 is the SERS spectrum of MGITC measured by the triangular column array SERS substrate at different etching times;

[0067] Figure 4 is the uniformity and reproducibility detection diagram of the triangular column array SERS substrate;

[0068] Figure 5 is the ultraviolet absorption spectrum of the gold-plated substrate, the modified aptamer, and the modified zwitterion substrate. DETAILED DESCRIPTION

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

[0070] Figure 1 is the preparation process diagram of the triangular column array substrate of the present application. Combined with Figure 1 The specific description is as follows:

[0071] First, the triangular column array is obtained by photolithography, and the microcolumn surface micro-nano structure is increased by femtosecond laser and inductively coupled plasma etching, so as to increase the specific surface area of the substrate, facilitate subsequent cell adhesion, and electron beam evaporate gold film on the substrate, so that the substrate can have more hot spots and have a better Raman signal enhancement effect.

[0072] The specific steps of the present application are as follows:

[0073] Step 1), preparation of the substrate stamp;

[0074] The preparation of the base stamp adopts ultraviolet lithography and reverse mold method. First, 30 μm SU-3035 photoresist is spin-coated on the cleaned silicon wafer, exposed for 40 s by a mask in a UV ultraviolet lithography machine, and exposed for about 13 s three times, then baked at 95℃ for about 2 min to make the pattern appear, and the base positive template is obtained by developing; PDMS and curing agent 10:1 are mixed, stirred uniformly, and degassed in a vacuum machine to remove bubbles, and the prepared positive template is injection molded, and the PDMS stamp is obtained by cutting with a knife in a 70℃ oven for 2 h;

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

[0076] A drop of SU-2010 photoresist is added on the glass sheet, and the stamp prepared in step 1) is pressed on the photoresist. After standing for 5 min, it is turned over and stood for another 5 min, so that the photoresist fills the stamp pattern. Finally, it is exposed in a UV box for 60 s. SU-2010 photoresist is used because it is relatively thin and can fill the stamp pattern.

[0077] Step 3), processing of the surface structure of the DLD triangular array base;

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

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

[0080] Figure 2 The SEM images of the base after stamping and curing, femtosecond laser ablation, and ICP etching are shown in the following table: Figure 2 As can be seen from the table, after laser ablation and ICP etching, the triangular prism side has better micro-nano structure, which is more conducive to improving the adsorption capacity of the base.

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

[0082] In order to make the base produce enhancement to the Raman signal and facilitate the modification of aptamer, 30 nm gold film is evaporated on the base processed in step 3) by electron beam vacuum coating instrument.

[0083] Step 5), SEM characterization of the triangular pyramid substrate;

[0084] In order to characterize the difference of the surface morphology of the substrate under femtosecond laser and different plasma etching time, the substrate under different processing conditions was pasted on the sample stage, the metal was sprayed to increase the conductivity of the substrate, and the surface morphology was recorded by scanning high-resolution electron microscope.

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

[0086] Figure 3 SERS spectra of MGITC measured for the triangular pyramid array SERS substrate under different etching time; Figure 4 The detection map of the uniformity and reproducibility of the triangular pyramid array SERS substrate; Figure 3 、 Figure 4 ,

[0087] The Raman signal of the substrate under different etching time was characterized to determine the optimal etching time of the substrate. The SERS substrate under different etching time was treated by oxygen plasma for about 2 min, immersed in 10 -6 mol / L MGITC solution for about 12 h, and Raman measurement was performed on the substrate under the microscope confocal Raman spectrometer. The uniformity and reproducibility of the Raman signal is an important indicator to measure the SERS performance.

[0088] The immersed substrate was taken out and air dried, 2.5-5 mW, integration time was 1 s, integration times was 1, 10 Raman spectra were measured under the Raman spectrometer, and the Raman intensity of the characteristic peak 1614 cm -1 of MGITC was taken, and the relative standard deviation was calculated to be 6.71%, which indicated that the uniformity of the substrate was good.

[0089] Step 7), substrate aptamer incubation;

[0090] The substrate modified aptamer is the basis for the substrate to capture CTCs. The best condition for gold plating substrate was treated in oxygen plasma cleaning machine for about two minutes, immersed in 70 nmol / L wy5a aptamer solution for 20-24 h, and the substrate was combined with the aptamer through Au-S bond. In order to make the substrate and the aptamer better combined, the honeycomb oscillator was used for shaking during soaking, and the free aptamer was removed by washing with PBS solution for 2-3 times.

[0091] Including step 8) zwitterionic synthesis and modification;

[0092] Zwitterion was synthesized by hydrogen peroxide oxidation of N,N-bis(3-aminopropyl)methylamine (DNMA) and modified on the substrate.

[0093] The specific implementation steps are as follows: 15ml 8% hydrogen peroxide solution is added dropwise in 20ml DNMA aqueous solution with a concentration of 34.5mmol / L under ice water bath, the last solution is stirred under ice water bath for 10-12h, the reaction completed solution is extracted with dichloromethane for 2-3 times, and DNMAO aqueous solution is obtained, and the aqueous solution is freeze-dried to obtain DNMAO solution. Meanwhile, the substrate modified with the aptamer in step 7) is placed in 1mmol / L 11-mercapto undecanoic acid tetrahydrofuran solution (11-MUA) for about 2h, 11MUA is combined with the substrate through AU-S, then the substrate is taken out and activated carboxyl in 1mmol / L 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mixed solution, then DNMAO synthesized is added dropwise on the substrate and reacts for about 2h, DNMAO is combined with carboxyl through condensation reaction, so as to realize modification on the substrate, and finally the substrate is washed with PBS for 1-2 times to remove uncombined molecules.

[0094] Figure 5 The UV absorption spectrum of the substrate in different modification stages is as follows:

[0095] The gold-plated substrate is taken, the aptamer is modified, the substrate modified with the aptamer and the zwitterion is cut to a proper size, and the absorption spectrum of the substrate in a wavelength range of 200-800nm is measured in the ultraviolet spectrophotometer, the modification process of the aptamer and the zwitterion is reflected through the spectrum change in different stages, so as to judge whether the aptamer and the zwitterion are modified on the substrate.

[0096] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and the present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a Raman enhanced substrate for resisting non-specific adsorption of organisms, characterized in that: The following steps are included: Step 1) preparing a base stamp by using UV lithography and a casting method; Draw the triangular prism base of the deterministic lateral displacement DLD using AutoCAD and print out a film version; A 30µm thick 3035 photoresist was spin-coated on the silicon wafer, and a positive template was prepared by photolithography; The base stamp is obtained by polydimethylsiloxane (PDMS) casting; Step 2), DLD triangular prism array substrate preparation; Place a drop of SU-2010 photoresist on a glass slide, press the stamp prepared in step 1) onto the photoresist, and photocuring is performed 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 subjected to femtosecond laser processing under optimized femtosecond laser parameters, and then the inductively coupled plasma is optimized to process micro-nano structures on the sidewalls of the triangular prisms to obtain a DLD triangular prism array substrate with micro-nano structures; Step 4), DLD triangular prism array substrate is gold plated; The processed substrate was coated with 30 nm of gold in an electron beam vacuum coating apparatus; Step 5), SEM characterization of DLD triangular prism array substrate; The surface morphology of 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 prepared in step 4) was subjected to oxygen plasma hydrophilization treatment and then placed in MGITC solution. The SERS performance uniformity of the substrate 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 then modified on a substrate.

2. The method for preparing a Raman enhanced substrate for resisting nonspecific adsorption of organisms according to claim 1, characterized in that: The step 1) is specifically as follows: First, a 30µm thick layer of SU-3035 photoresist was spin-coated on a cleaned silicon wafer. The mask was exposed for 40 seconds using a UV lithography machine, with three exposures of 13 seconds each. The pattern was then post-baked at 95°C for 2 minutes to reveal the pattern. A positive template of the substrate was obtained through development. PDMS and a curing agent were mixed in a 10:1 ratio and stirred evenly. The mixture was then vacuum-extracted to remove air bubbles. The prepared positive template was then injection-molded and cured in a 70°C oven for 1-2 hours. The PDMS stamp was then cut using a cutter. The triangular micropillars prepared on the triangular prism substrate of the deterministic lateral displacement (DLD) have a height of 30µm, a side length of 40 microns, an offset angle of 3-5°, and a critical diameter of 15µm.

3. The method for preparing a Raman enhanced substrate for resisting nonspecific adsorption of organisms according to claim 1, characterized in that: The step 2) is specifically as follows: Place a drop of SU-2010 photoresist on a glass slide and press the stamp made in step 1) onto the photoresist. Let it sit for 5 minutes, then flip it over and let it sit for another 5 minutes to allow the photoresist to fill with the stamp pattern. Finally, expose it in a UV box for 60-120 seconds.

4. The method for preparing a Raman enhanced substrate for resisting nonspecific 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: The substrate processed in step 2) is processed by a femtosecond laser with an operating power of 18-32 mW, a frequency of 5 kHz, and a scanning speed of 5 mm / s 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 150W, lower radio frequency 50W, oxygen flow rate set to 30sccm, helium 20sccm, and etching time 7.5min; Thus, micro-nano structures are etched on the sidewalls.

5. The method for preparing a Raman enhanced substrate for resisting nonspecific 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, then air-dried for testing; The Raman spectrometer used was a Renishaw Invia confocal Raman spectrometer. The spectrometer parameters were set as follows: excitation light source of 633 nm, 50× microscope objective, laser power of 2.5–5 mW, integration time of 1 s, and integration times of 1.

6. The method for preparing a Raman enhanced substrate for resisting nonspecific adsorption of organisms 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; Prepare a pre-prepared 70 nmol / L specific aptamer solution, then immerse the treated substrate in the aptamer solution and hang it on the edge of a honeycomb oscillator for 20-24 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 for resisting nonspecific adsorption of organisms 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 10-12 hours. Extract the reaction solution with dichloromethane 2-3 times to obtain DNMAO. Soak the substrate modified with the aptamer in step 7) in a 1 mmol / L 11-mercaptoundecanoic acid solution for 1-2 hours. Then, remove the substrate and activate the carboxyl group in a 1 mmol / L mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Then, add the synthesized DNMAO dropwise onto the substrate and react for 1-2 hours. Finally, wash the substrate with PBS solution 1-2 times to remove unbound molecules.

8. Use of a substrate prepared by the method for preparing a substrate for anti-biological nonspecific adsorption Raman enhancement according to any one of claims 1 to 7 in a microfluidic chip.

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

Images