Preparation method and application of SERS substrate based on F-P cavity

Abstract

The invention discloses a preparation method of an SERS (Surface Enhanced Raman Scattering) substrate based on an F-P (Fabry-Perot) cavity, preparation and application of the SERS substrate based on the F-P cavity prepared by the method in a micro-fluidic chip, and application of the micro-fluidic chip adopting the SERS substrate based on the F-P cavity in capturing and detecting circulating tumor cells. The preparation method of the substrate comprises the following basic steps: S1, selecting a PET film as a supporting layer; and S2, preparing the SERS substrate with an F-P cavity with a micron array on the surface on the PET film. Raman scattering is increased by fusing light interference enhanced Raman scattering of a Fabry-Perot cavity and rough precious metal surface enhanced Raman scattering, and the micro-nano array is further arranged on the surface of the SERS substrate, so that collision between circulating tumor cells and the micro-nano array can be effectively increased, the speed of the circulating tumor cells is slowed down, and the service life of the circulating tumor cells is prolonged. The invention provides a new method for capturing and detecting the circulating tumor cells, and has great significance for early screening and diagnosis of human prostate cancer and heterogeneity analysis and research of the circulating tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of micro-nano sensing technology, and in particular, relates to a preparation method and application of a SERS substrate based on an FP cavity. Background Art

[0002] In 1928, physicist CV Raman discovered that the spectrum of each substance is unique, similar to a human fingerprint, and can distinguish specific molecules in a sample. However, due to its low sensitivity, its intensity is only 10 times that of the incident light intensity. -6 ~10 -10 , this intensity cannot be used to study the structure and properties of molecules, and surface enhanced Raman scattering (SERS) can make up for this deficiency. SERS refers to when some molecules are adsorbed onto certain rough metal surfaces, their Raman scattering intensity will increase by 10 4 ~10 12 times, thereby enhancing the Raman signal.

[0003] The Fabry-Perot cavity is an optical cavity composed of a thin metal layer on the top layer, a thick metal layer on the bottom layer, and a transparent medium layer in the middle. At this stage, it has been proposed and proven that the use of the Fabry-Perot cavity as a Raman substrate can obtain about an order of magnitude of signal enhancement from light interference. In this design, the incident wave and the reflected wave combine to form multi-beam interference, generating a standing wave in the electric field, thereby enhancing the electromagnetic field and thus the intensity of Raman scattering. When the incident light passes through the probe molecule layer, it is partially absorbed by the reporter molecule, causing the light to be reflected multiple times in the cavity, giving the light multiple opportunities to be absorbed by the probe molecule layer. The signal enhancement comes from constructive interference in the propagating light wave. This type of far-field optical enhancement does not rely on the enhancement of plasmon resonance and molecular resonance, but is applied in parallel to plasmon resonance and molecular resonance.

[0004] Microfluidics refers to the science and technology involved in systems that use microchannels (tens to hundreds of microns in size) to process or manipulate tiny fluids (with volumes ranging from picoliters to nanoliters).

[0005] Microfluidic chips are a scientific technology that can manipulate fluids in micrometer-scale space. This technology has the ability to concentrate the basic functions of biological and chemical laboratories on a chip of less than a few square centimeters. Most of the current mainstream microfluidic chips are formed by microchannels to form a network, and use controllable fluids to run through the entire chip to achieve various functions required for conventional chemical or biological experiments. Its basic characteristics and greatest advantages are to use a variety of microstructure units on a tiny controllable platform to cleverly combine and integrate them on a large scale to complete detection, analysis and other functions.

[0006] In the prior art, most of the processing techniques in the SERS substrate manufacturing process are high in cost and demanding in processing conditions, and the SERS detection sensitivity is relatively insufficient.

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to solve the problems of complex processing conditions in the process of preparing traditional SERS substrates for capturing and detecting circulating tumor cells, and provides a method for preparing a SERS substrate based on an FP cavity, and the preparation and application of the SERS substrate based on the FP cavity prepared by the method in a microfluidic chip, and the application of the microfluidic chip using the SERS substrate based on the FP cavity in the capture and detection of circulating tumor cells.

[0009] To achieve the above purpose, the technical solution provided by the present invention is:

[0010] A method for preparing a SERS substrate based on a FP cavity specifically comprises the following steps:

[0011] Step S1, selecting a PET film as a support layer;

[0012] Select a PET film with a thickness of 100 to 150 μm as the support layer of the FP cavity, clean the PET film with 75% alcohol and dry it to complete the preparation work;

[0013] Step S2, preparing a SERS substrate having a micron array of FP cavities on the surface on a PET film;

[0014] Step S2.1, preparing the bottom layer of the FP cavity;

[0015] The bottom layer of the FP cavity is prepared by sequentially coating a silver film and a gold film on the PET film described in step S1 through an electron beam vacuum coating process;

[0016] Step S2.2, preparing a micro-nano array;

[0017] Spin-coat a photoresist on the bottom layer of the FP cavity prepared in step S2.1 and perform a photolithography operation to prepare a micrometer array, thereby obtaining a bottom layer of the FP cavity with a micrometer array on the surface;

[0018] Step S2.3, preparing the middle transparent dielectric layer of the FP cavity;

[0019] Spin-coat PMMA on the FP cavity bottom layer with the micron array on the surface prepared in step S2.2 to form a transparent medium thin layer on the bottom layer;

[0020] Step S2.4, preparing the top layer of the FP cavity;

[0021] Repeat step S2.1 to form a top layer by coating a gold film again using an electron beam vacuum coating process;

[0022] The preparation of the SERS substrate with a micron array of FP cavities on the surface is completed.

[0023] The preferred technical solution provided by the present invention is:

[0024] The step S2.1 is specifically as follows:

[0025] Put the cleaned PET film in step S1 into the electron beam vacuum coating instrument, open the protective baffle, turn on the molecular pump of the coating instrument to evacuate to 5.0×10 -4 When the voltage is below 100V, turn on the relay, filament and high voltage power supply successively, and slowly adjust the voltage to 89V-93V. When the coating rate rises to / min and stabilize, close the protective baffle and start coating;

[0026] When the coating reaches 170nm, open the protective baffle, turn off the high voltage and filament relays successively to stop coating and take out;

[0027] Debug the electron beam vacuum coating instrument and adjust the voltage to about 92V until the coating rate stabilizes. / min or so, start gold plating, open the protective baffle when the plating reaches 30nm, turn off the high voltage and filament relays successively to stop plating, and take out;

[0028] Complete the bottom layer preparation of the FP cavity.

[0029] The preferred technical solution provided by the present invention is:

[0030] The step S2.2 is specifically as follows:

[0031] Step S2.2.1, pre-bake the bottom layer of the FP cavity prepared in step S2.1 on a heating plate set at a temperature of 105° for 10 minutes, then place it on a glue spreader and turn on the vacuum pump to suck it tightly;

[0032] Step S2.2.2, pouring photoresist Su-3035 on the bottom layer of step S2.2.1, and operating a coating machine to perform a coating spinning process to obtain a layer of photoresist with a thickness of 30 microns on the bottom layer;

[0033] Step S2.2.3, place the FP cavity bottom layer of step S2.2.2 on a heating plate at 95° for 10 minutes, then place it on the UV lithography machine exposure table and suck it tightly, and stick the designed and prepared mask on the glass sheet and align it with the sample on the exposure table for exposure;

[0034] Step S2.2.4, place the bottom layer of the FP cavity in step S2.2.3 on a heating plate at 95°C and bake for 10 min;

[0035] Step S2.2.5, pouring ethyl lactate solution into the glass culture dish, ensuring that the liquid surface of ethyl lactate can cover the silicon wafer, and then placing the bottom layer after post-baking into ethyl lactate for development;

[0036] Step S2.2.6, use a nitrogen gun to gently blow dry the developer on the surface of the silicon wafer, and complete the preparation of the FP cavity bottom layer with a micron array. The preferred technical solution provided by the present invention is:

[0037] The step S2.3 is specifically as follows:

[0038] Mix PMMA powder and ethyl lactate in a ratio of 1:7 by weight and stir magnetically for about 12 hours until it becomes transparent and free of bubbles;

[0039] The PET with micron array on the bottom layer of the FP cavity after coating is placed on the glue machine and sucked tightly;

[0040] Use a rubber-tipped dropper to take out 3 ml to 4 ml of the prepared 1:7 PMMA transparent solution and drop it on the bottom layer of the FP cavity with the micron array, and spin-coat it on the bottom layer to form the middle transparent medium layer of the FP cavity.

[0041] The present invention also provides a technical solution for preparing and applying a SERS substrate based on an FP cavity in a microfluidic chip.

[0042] The present invention also provides a technical solution for the application of a SERS substrate based on a FP cavity in the capture of circulating tumor cells. The specific steps include:

[0043] Step S3, modifying two specific aptamers on the SERS substrate;

[0044] First, the prepared SERS substrate is subjected to hydrophilic treatment, and then the treated SERS substrate is placed in a wy-5a and PSMA aptamer solution, slowly shaken for 24 hours, and finally the residual aptamer that has not been modified is washed away with a buffer solution to complete the modification of the substrate;

[0045] Step S4, capturing circulating tumor cells;

[0046] The SERS substrate modified with specific antibodies in step S3 is placed in the capture area of ​​the microfluidic chip, and the circulating tumor cells are pretreated to be fluorescently stained. Two types of circulating tumor cells are taken and introduced into the microfluidic chip entrance along with the suspension. After the introduction, they are rinsed with buffer and the SERS substrate is observed under a fluorescence microscope to capture the circulating tumor cells. Finally, the cells are dehydrated, fixed, etc., and the cells are observed under SEM to see whether they extend pseudopodia and are well captured by the SERS substrate.

[0047] Preferably,

[0048] The step S4 specifically includes:

[0049] Step S4.1, place the suspension of the two circulating tumor cells in a dark, light-proof centrifuge tube, add 10 μL of AO / EB mixture, and place it in a dark environment at room temperature for staining for 10-15 minutes to complete cell staining;

[0050] Step S4.2, the stained circulating tumor cells and the suspension are sucked into a syringe, the outlet of the syringe is connected to the inlet of the microfluidic chip with a hose, the SERS substrate is embedded in the capture area of ​​the microfluidic chip, the parameters of the automatic syringe pusher are set to 10 μL / min, and the syringe is pushed to allow the cell suspension to flow into the microfluidic chip to reach the SERS substrate in the capture area;

[0051] Step S4.3, after the cell suspension is passed, PBS buffer is passed through another inlet of the microfluidic chip at a flow rate of 2 μL / min to flush away the circulating tumor cells that have not been adsorbed on the SERS substrate;

[0052] Step S4.4, observing the capture situation using a fluorescence microscope;

[0053] Step S4.5, adding 30 μL of 2.5% glutaraldehyde solution to the substrate for fixation for 2-3 hours; glutaraldehyde is used to fix cancer cells and maintain their morphology when captured;

[0054] Step S4.6, placing the SERS substrate in alcohol solutions with concentrations of 30%, 50%, and 80% for 2 minutes each;

[0055] Step S4.7, attach the SERS substrate to a sample table with an inclination angle of 45 degrees and spray palladium for 120 to 150 seconds;

[0056] The cells captured by the SERS substrate can then be observed using SEM.

[0057] Wherein, the circulating tumor cells refer to PC-3 tumor cells and LNCaP tumor cells in prostate cancer.

[0058] The present invention also provides an application of a SERS substrate based on an FP cavity in the detection of circulating tumor cells.

[0059] The method further includes performing SERS detection on the prepared SERS substrate of the Burli-Perot cavity as described in step S5, wherein the specific operation is as follows:

[0060] The SERS substrate was treated with oxygen plasma for 90-120 s and then soaked in 10 -6mol / L MGITC solution, protected from light for 3-4h, finally taken out and rinsed with deionized water to remove the free MGITC Raman reporter molecules not attached to the SERS substrate, and placed in a petri dish to air dry naturally. The prepared SERS substrate was placed on the test sample stage of the Renishaw Raman spectrometer, and then the SERS substrate was Raman detected and the results of the statistical test were collected. The test parameters of the Renishaw Raman spectrometer were: 20× microscope objective, excitation light source: 633nm, laser power: 1.7mW-5.5mW, integration time 1s, integration times 1 time, laser intensity 5%, and data from 10 random points on the SERS substrate were collected and recorded, and the average value was generated to compare the Raman signal with that of the ordinary gold-plated substrate.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The present invention solves the problem of complex processing conditions in the process of preparing traditional SERS substrates for capturing and detecting circulating tumor cells, and provides a new idea, that is, integrating the optical interference enhanced Raman scattering of the Fabry-Perot cavity and the rough noble metal surface enhanced Raman scattering to increase Raman scattering. In addition, there is a micro-nano array on the surface of the SERS substrate, which can effectively increase the collision between the circulating tumor cells and the micro-nano array, slow down the speed of the circulating tumor cells, so that the specific aptamer modified on the surface of the substrate can well capture the circulating tumor cells. The invention can be mass-produced, has high repeatability, is simple to prepare, and does not require processing with high-risk equipment. The prepared substrate has good Raman scattering enhancement effect, strong stability, is non-toxic and harmless to cells, and has little damage, and can be used for capturing and detecting circulating tumor cells.

[0063] Terminology explanation:

[0064] FP cavity——abbreviation of Fabry-Perot cavity;

[0065] PET – Polyethylene terephthalate;

[0066] PDMS – polydimethylsiloxane;

[0067] PGMEA – Propylene glycol methyl ether acetate;

[0068] MGITC – Malachite green isothiocyanate solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Schematic diagram of the overall structure of the SERS substrate based on the FP cavity of the present invention;

[0070] Figure 2 It is a schematic diagram of the preparation process of the SERS substrate based on the FP cavity of the present invention;

[0071] Figure 3 for Figure 2 Schematic diagram of the process of forming micrometer arrays by mid-UV lithography;

[0072] Figure 4 The actual image (left) and SEM image (right) of the prepared SERS substrate;

[0073] Figure 5 SEM images of circulating tumor cells captured by substrate;

[0074] Figure 6 Raman signal detection for the prepared SERS substrate;

[0075] Figure 7 To detect the uniformity of the prepared SERS substrate. DETAILED DESCRIPTION

[0076] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings.

[0077] Figure 1 Schematic diagram of the overall structure of the SERS substrate based on the FP cavity of the present invention; Figure 2 It is a schematic diagram of the preparation process of the SERS substrate based on the FP cavity of the present invention; Figure 3 for Figure 2 Schematic diagram of the process of forming micrometer arrays by medium ultraviolet lithography; Figure 1 , Figure 2 as well as Figure 3 As shown in the figure, a method for preparing a SERS substrate based on a FP cavity of the present invention comprises the following steps:

[0078] Step S1, selecting a PET film as a support layer;

[0079] Select a PET film with a thickness of 100 to 150 μm as the support layer of the FP cavity, clean the PET film with 75% alcohol and dry it to complete the preparation work;

[0080] The PET is polyethylene terephthalate, and its chemical formula is (C10H8O4)n. A PET film with a thickness of 100-150 μm is selected as the support layer of the SERS substrate. The film is rinsed with deionized water and 75% alcohol, dried, and then used for later use.

[0081] PET is a relatively transparent polymer with a smooth surface, good creep resistance, fatigue resistance, abrasion resistance and dimensional stability, high hardness, the greatest toughness among thermoplastics, little affected by temperature, non-toxic, weather-resistant, good chemical resistance, low water absorption, and resistance to weak acids and organic solvents.

[0082] Prepare 75% alcohol and spray it on the surface of PET, wipe it with a soft dust-free cloth and place it on a heating plate to dry. This step is to remove the residual organic solvents and tiny debris on the surface of PET;

[0083] Step S2, preparing a SERS substrate having a micron array of FP cavities on the surface on a PET film;

[0084] Step S2.1, preparing the bottom layer of the FP cavity;

[0085] The bottom layer of the FP cavity is prepared by sequentially coating a silver film and a gold film on the PET film described in step S1 through an electron beam vacuum coating process;

[0086] The step S2.1 is as follows: put the cleaned PET film in step S1 into the electron beam vacuum coating apparatus, open the protective baffle, turn on the molecular pump of the coating apparatus to evacuate the film to 5.0×10 -4 When the voltage is below 100V, turn on the relay, filament and high voltage power supply successively, and slowly adjust the voltage to 89V-93V. When the coating rate rises to / min and stabilize, close the protective baffle and start coating;

[0087] When the coating reaches 170nm, open the protective baffle, turn off the high voltage and filament relays successively to stop coating and take out;

[0088] Debug the electron beam vacuum coating instrument and adjust the voltage to about 92V until the coating rate stabilizes. / min or so, start gold plating, open the protective baffle when the plating reaches 30nm, turn off the high voltage and filament relays successively to stop plating, and take out;

[0089] Complete the bottom layer preparation of the FP cavity.

[0090] Step S2.2, preparing a micro-nano array;

[0091] Spin-coat a photoresist on the bottom layer of the FP cavity prepared in step S2.1 and perform a photolithography operation to prepare a micrometer array, thereby obtaining a bottom layer of the FP cavity with a micrometer array on the surface;

[0092] The step S2.2 is specifically as follows:

[0093] Step S2.2.1, pre-bake the bottom layer of the FP cavity prepared in step S2.1 on a heating plate set at a temperature of 105° for 10 minutes, then place it on a glue spreader and turn on the vacuum pump to suck it tightly;

[0094] Step S2.2.2, pour the photoresist Su-3035 on the bottom layer of step S2.2.1, operate the coating machine to perform the coating process to obtain a layer of photoresist with a thickness of 30 microns on the bottom layer; (the parameters of the coating machine are: overall acceleration 300rpm / s, first at 500rpm / s for 18s, and then at 3500rpm / s for 60s).

[0095] Step S2.2.3, place the bottom layer of the FP cavity in step S2.2.2 on a 95° heating plate and bake for 10 minutes, then place it on the UV lithography exposure table and suck it tightly, stick the designed and prepared mask on the glass sheet and align it with the sample on the exposure table for exposure, the UV lithography power is 19.2mW*cm -2 ,The total exposure time is 39s, divided into three times, 13s each, with an interval of 10s;

[0096] Step S2.2.4, place the bottom layer of the FP cavity in step S2.2.3 on a heating plate at 95°C and bake for 10 min;

[0097] Step S2.2.5, pour ethyl lactate solution into the glass culture dish, make sure the liquid surface of ethyl lactate can cover the silicon wafer, and then put the bottom layer after post-baking into ethyl lactate for development; this step is to remove the uncross-linked photoresist, after developing for 5 minutes, drip two drops of isopropyl alcohol on the pattern, because isopropyl alcohol can react with the photoresist to generate white flocs. If it is not developed cleanly, white flocs will appear, and continue to develop until no white flocs appear;

[0098] Step S2.2.6, use a nitrogen gun to gently blow dry the developer on the surface of the silicon wafer to complete the preparation of the FP cavity bottom layer with the micron array.

[0099] Step S2.3, preparing the middle transparent dielectric layer of the FP cavity;

[0100] Spin-coat PMMA on the FP cavity bottom layer with the micron array on the surface prepared in step S2.2 to form a transparent medium thin layer on the bottom layer;

[0101] The step S2.3 is specifically as follows:

[0102] Mix PMMA powder and ethyl lactate in a ratio of 1:7 by weight and stir magnetically for about 12 hours until it becomes transparent and free of bubbles;

[0103] The PET with micron array on the bottom layer of the FP cavity after coating is placed on the glue machine and sucked tightly;

[0104] Use a rubber-tipped dropper to take out 3ml to 4ml of the prepared 1:7 PMMA transparent solution and drop it on the bottom layer of the FP cavity with the micron array, and spin-coat it on the bottom layer to form the middle transparent medium layer of the FP cavity. (The operating parameters of the coating machine: acceleration 300rpm / s, 1500rpm / s for 15s; 8000rpm / s for 95s);

[0105] Step S2.4, preparing the top layer of the FP cavity;

[0106] Repeat step S2.1 to form a top layer by coating a gold film again using an electron beam vacuum coating process;

[0107] The preparation of the SERS substrate with a micron array of FP cavities on the surface is completed.

[0108] The operation steps for debugging the electron beam vacuum coating apparatus are the same as S2.1. Adjust the voltage to about 92V and wait for the coating rate to stabilize. / min or so, the gold film was plated, and when the plated thickness reached 30nm, the protective baffle was opened, and the high voltage, filament, and relay were turned off successively to stop the film plating. So far, the FP cavity substrate with a micron array structure has been prepared.

[0109] Figure 4 The left picture shows the actual picture of the prepared FP cavity SERS substrate with a micron array structure. Figure 4 The middle right picture is a SEM picture.

[0110] The SERS substrate based on the FP cavity of the present invention can be used to prepare a microfluidic chip, and the prepared microfluidic chip can be used for capturing or detecting circulating tumor cells.

[0111] The circulating tumor cells refer to PC-3 tumor cells and LNCaP tumor cells in prostate cancer.

[0112] in,

[0113] The application of the SERS substrate based on the FP cavity in the capture of circulating tumor cells includes the following specific steps:

[0114] Step S3, modifying two specific aptamers on the SERS substrate;

[0115] First, the prepared SERS substrate is subjected to hydrophilic treatment, and then the treated SERS substrate is placed in a wy-5a and PSMA aptamer solution, slowly shaken for 24 hours, and finally the residual aptamer that has not been modified is washed away with a buffer solution to complete the modification of the substrate;

[0116] In step S3, two specific aptamers are modified on the SERS substrate, and the substrate can capture two circulating tumor cells at the same time. Compared with the traditional single circulating tumor cell detection, the results are more reliable. The two circulating tumor cells PC-3 and LNCaP are epithelial and mesenchymal, respectively, which provides help for the heterogeneity analysis of cancer cells.

[0117] Step S4, capturing circulating tumor cells;

[0118] Figure 5 The SEM image of cells captured by SERS substrate is as follows: Figure 4 As shown in .

[0119] The SERS substrate captures circulating tumor cells and takes SEM photos. The SERS substrate modified with specific antibodies in step S3 is placed in the capture area of ​​the microfluidic chip. The circulating tumor cells are pretreated and fluorescently stained. Two types of circulating tumor cells are taken and introduced into the microfluidic chip entrance along with the suspension. After the introduction, they are rinsed with buffer and the SERS substrate is observed under a fluorescence microscope for the capture of circulating tumor cells. Finally, the cells are dehydrated, fixed, etc., and observed under SEM to see whether the cells extend pseudopodia and are well captured by the SERS substrate.

[0120] The step S4 specifically includes:

[0121] Step S4.1, place the suspension of the two circulating tumor cells in a dark, light-proof centrifuge tube, add 10 μL of AO / EB mixture, and place it in a dark environment at room temperature for staining for 10-15 minutes to complete cell staining;

[0122] Step S4.2, the stained circulating tumor cells and the suspension are sucked into a syringe, the outlet of the syringe is connected to the inlet of the microfluidic chip with a hose, the SERS substrate is embedded in the capture area of ​​the microfluidic chip, the parameters of the automatic syringe pusher are set to 10 μL / min, and the syringe is pushed to allow the cell suspension to flow into the microfluidic chip to reach the SERS substrate in the capture area;

[0123] Step S4.3, after the cell suspension is passed, PBS buffer is passed through another inlet of the microfluidic chip at a flow rate of 2 μL / min to flush away the circulating tumor cells that have not been adsorbed on the SERS substrate;

[0124] Step S4.4, observing the capture status with a fluorescence microscope; Step S4.5, adding 30 μL of 2.5% glutaraldehyde solution to the substrate for fixation for 2-3 hours; glutaraldehyde is used to fix cancer cells to maintain their morphology when captured;

[0125] Step S4.6, placing the SERS substrate in alcohol solutions with concentrations of 30%, 50%, and 80% for 2 minutes each;

[0126] Step S4.7, attach the SERS substrate to a sample table with an inclination angle of 45 degrees and spray palladium for 120 to 150 seconds;

[0127] The cells captured by the SERS substrate can then be observed using SEM.

[0128] Figure 6 To detect the Raman signal and uniformity of the prepared SERS substrate;

[0129] The SERS substrate was treated with hydrophilicity, and then immersed in the Raman reporter molecule MGITC solution for 2 hours, and then taken out and rinsed with deionized water to remove the residual Raman reporter molecules. Finally, Raman detection was performed.

[0130] Step 3 is as follows:

[0131] The SERS substrate was treated with oxygen plasma for 90-120 s and then soaked in 10 -6 mol / L MGITC solution in the dark for 3-4 h, take it out with tweezers, rinse it with deionized water to remove the free MGITC Raman reporter molecules not attached to the SERS substrate, and place it in a culture dish to air dry naturally.

[0132] The prepared SERS substrate was placed on the test sample stage of the Renishaw Raman spectrometer, and the SERS substrate was tested by the Renishaw Raman spectrometer system. The test parameters of the Renishaw Raman spectrometer were: 20× microscope objective, excitation: 633nm HeNe laser, laser power: 1.7mW~5.5mW, integration time 1s, integration times 1 time, laser intensity 5%. The Raman signal data of 10 random points on the SERS substrate were collected and recorded, the uniformity of SERS was detected and the average value was generated as the final result of the Raman signal of the prepared SERS substrate. The Raman signal of the ordinary gold-plated substrate was compared. The results show that the gold-plated substrate with FP cavity has a better Raman enhancement effect. Figure 6 As shown, circulating tumor cells can also be captured. Figure 5 As shown in the figure, this is something that a smooth gold-plated substrate cannot do. The uniformity of SERS signals is one of the key indicators of SERS substrates. SERS signals are detected at 10 random points on the same SERS substrate. Generally speaking, if the RSD (relative standard deviation) is below 10%, it means that the uniformity is good. Figure 7 As shown, the prepared SERS substrate was characterized ten times by the peak value of the characteristic peak 1614 cm-1 of the Raman reporter molecule MGITC to measure the uniformity of the substrate, RSD≈8.74%, and the uniformity was good.

[0133] 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 SERS substrate based on a FP cavity, characterized in that: The specific steps include: Step S1, selecting a PET film as a support layer; Select a PET film with a thickness of 100 to 150 μm as the support layer of the FP cavity, clean the PET film with 75% alcohol and dry it to complete the preparation work; Step S2, preparing a SERS substrate having a micron array of FP cavities on the surface on a PET film; Step S2.1, preparing the bottom layer of the FP cavity; The bottom layer of the FP cavity is prepared by sequentially coating a silver film and a gold film on the PET film described in step S1 through an electron beam vacuum coating process; Step S2.2, preparing a micro-nano array; Spin-coat a photoresist on the bottom layer of the FP cavity prepared in step S2.1 and perform a photolithography operation to prepare a micrometer array, thereby obtaining a bottom layer of the FP cavity with a micrometer array on the surface; Step S2.3, preparing the middle transparent dielectric layer of the FP cavity; Spin-coat PMMA on the FP cavity bottom layer with the micron array on the surface prepared in step S2.2 to form a transparent medium thin layer on the bottom layer; Step S2.4, preparing the top layer of the FP cavity; Repeat step S2.1 to form a top layer by coating a gold film again using an electron beam vacuum coating process; The preparation of the SERS substrate with a micron array of FP cavities on the surface is completed.

2. The method for preparing a SERS substrate based on a FP cavity according to claim 1, characterized in that: The step S2.1 is specifically as follows: Put the cleaned PET film in step S1 into the electron beam vacuum coating instrument, open the protective baffle, turn on the molecular pump of the coating instrument to evacuate to 5.0×10 -4 When the voltage is below 100V, turn on the relay, filament and high voltage power supply successively, and slowly adjust the voltage to 89V-93V. When the coating rate rises to When it is stable, close the protective baffle and start coating; When the coating reaches 170nm, open the protective baffle, turn off the high voltage and filament relays successively to stop coating and take out; Debug the electron beam vacuum coating instrument and adjust the voltage to about 92V until the coating rate stabilizes. When the gold film is plated to about 30nm, open the protective baffle, turn off the high voltage and filament relays to stop the film plating, and take it out; Complete the bottom layer preparation of the FP cavity.

3. The method for preparing a SERS substrate based on a FP cavity according to claim 1, characterized in that: The step S2.2 is specifically as follows: Step S2.2.1, pre-bake the bottom layer of the FP cavity prepared in step S2.1 on a heating plate set at a temperature of 105° for 10 minutes, then place it on a glue spreader and turn on the vacuum pump to suck it tightly; Step S2.2.2, pouring photoresist Su-3035 on the bottom layer of step S2.2.1, and operating a coating machine to perform a coating spinning process to obtain a layer of photoresist with a thickness of 30 microns on the bottom layer; Step S2.2.3, place the FP cavity bottom layer of step S2.2.2 on a heating plate at 95° for 10 minutes, then place it on the UV lithography machine exposure table and suck it tightly, and stick the designed and prepared mask on the glass sheet and align it with the sample on the exposure table for exposure; Step S2.2.4, place the bottom layer of the FP cavity in step S2.2.3 on a heating plate at 95°C and bake for 10 min; Step S2.2.5, pouring ethyl lactate solution into the glass culture dish, ensuring that the liquid surface of ethyl lactate can cover the silicon wafer, and then placing the bottom layer after post-baking into ethyl lactate for development; Step S2.2.6, use a nitrogen gun to gently blow dry the developer on the surface of the silicon wafer to complete the preparation of the FP cavity bottom layer with the micron array.

4. The method for preparing a SERS substrate based on a FP cavity according to claim 1, characterized in that: The step S2.3 is specifically as follows: Mix PMMA powder and ethyl lactate in a ratio of 1:7 by weight and stir magnetically for about 12 hours until it becomes transparent and free of bubbles; The PET with micron array on the bottom layer of the FP cavity after coating is placed on the glue machine and sucked tightly; Use a rubber-tipped dropper to take out 3 ml to 4 ml of the prepared 1:7 PMMA transparent solution and drop it on the bottom layer of the FP cavity with the micron array, and spin-coat it on the bottom layer to form the middle transparent medium layer of the FP cavity.

5. A method for preparing and applying the SERS substrate based on the FP cavity according to any one of claims 1 to 4 in a microfluidic chip.

6. Use of the SERS substrate based on the FP cavity according to any one of claims 1 to 4 in capturing circulating tumor cells.

7. The use of the SERS substrate based on the FP cavity in the capture of circulating tumor cells according to claim 6, characterized in that: The specific steps include: Step S3, modifying two specific aptamers on the SERS substrate; First, the prepared SERS substrate is subjected to hydrophilic treatment, and then the treated SERS substrate is placed in a wy-5a and PSMA aptamer solution, slowly shaken for 24 hours, and finally the residual aptamer that has not been modified is washed away with a buffer solution to complete the modification of the substrate; Step S4, capturing circulating tumor cells; The SERS substrate modified with specific antibodies in step S3 is placed in the capture area of ​​the microfluidic chip, and the circulating tumor cells are pretreated to be fluorescently stained. Two types of circulating tumor cells are taken and introduced into the microfluidic chip entrance along with the suspension. After the introduction, they are rinsed with buffer and the SERS substrate is observed under a fluorescence microscope to capture the circulating tumor cells. Finally, the cells are dehydrated, fixed, etc., and the cells are observed under SEM to see whether they extend pseudopodia and are well captured by the SERS substrate.

8. The use of the SERS substrate based on the FP cavity in the capture of circulating tumor cells according to claim 7, characterized in that: The step S4 specifically includes: Step S4.1, place the suspension of the two circulating tumor cells in a dark, light-proof centrifuge tube, add 10 μL of AO / EB mixture, and place it in a dark environment at room temperature for staining for 10-15 minutes to complete cell staining; Step S4.2, the stained circulating tumor cells and the suspension are sucked into a syringe, the outlet of the syringe is connected to the inlet of the microfluidic chip with a hose, the SERS substrate is embedded in the capture area of ​​the microfluidic chip, the parameters of the automatic syringe pusher are set to 10 μL / min, and the syringe is pushed to allow the cell suspension to flow into the microfluidic chip to reach the SERS substrate in the capture area; Step S4.3, after the cell suspension is passed, PBS buffer is passed through another inlet of the microfluidic chip at a flow rate of 2 μL / min to flush away the circulating tumor cells that have not been adsorbed on the SERS substrate; Step S4.4, observing the capture situation using a fluorescence microscope; Step S4.5, adding 30 μL of 2.5% glutaraldehyde solution to the substrate for fixation for 2-3 hours; glutaraldehyde is used to fix cancer cells and maintain their morphology when captured; Step S4.6, placing the SERS substrate in alcohol solutions with concentrations of 30%, 50%, and 80% for 2 minutes each; Step S4.7, attach the SERS substrate to a sample table with an inclination angle of 45 degrees and spray palladium for 120 to 150 seconds; The cells captured by the SERS substrate can then be observed using SEM.

9. The use of the SERS substrate based on the FP cavity in the capture of circulating tumor cells according to claim 7, characterized in that: The circulating tumor cells refer to PC-3 tumor cells and LNCaP tumor cells in prostate cancer.

10. Use of the SERS substrate based on the FP cavity according to any one of claims 1 to 4 in the detection of circulating tumor cells.

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