Preparation method and application of graded micro-bowl inner cone array SERS (Surface Enhanced Raman Scattering) substrate

Abstract

The invention discloses a preparation method and application of a graded micro-bowl inner cone array SERS (Surface Enhanced Raman Scattering) substrate, and belongs to the technical field of surface enhanced Raman spectrum detection and microfluidics. The preparation method comprises the following steps: S1, self-assembling single-layer PS microspheres on PET (Polyethylene Terephthalate); s2, carrying out reverse molding on the photoresist to form a micro-bowl array; s3, the single-layer PS nanospheres are self-assembled in a bowl; s4, performing ICP etching on the nanospheres to form a bowl inner cone array; s5, a layer of gold film is formed through electron beam evaporation; s6, carrying out substrate characterization; s7, functionalizing the substrate; s8, preparing a micro-fluidic chip and sealing the micro-fluidic chip with an SERS substrate; and S9, heavy metal ion trace detection. The graded micro-bowl inner cone array SERS substrate provided by the invention is stable in property, not easy to oxidize, uniform in structure, large in specific surface area and rich in hot spots, and the micro-nano graded structure of the graded micro-bowl inner cone array SERS substrate can better regulate and control an incident electromagnetic light field, so that the Raman scattering effect is enhanced, and the graded micro-bowl inner cone array SERS substrate has great significance in trace detection of an object to be detected.

Description

Technical Field

[0001] The present invention belongs to the field of surface enhanced Raman spectroscopy detection and microfluidics technology, and in particular, relates to a preparation method of a graded micro-bowl inner cone array SERS substrate and its application. Background Art

[0002] Polluted heavy metals can accumulate in organisms and spread through the food chain, which reduces the diversity and richness of aquatic species and leads to the degradation of aquatic ecosystems. Their non-biodegradability, persistence and biomagnification pose a serious threat to human health. According to the tolerable intake data of different heavy metals published by the World Health Organization, the daily tolerable intake of mercury and lead is less than 1μg / kg / day. In particular, heavy metal mercury is extremely toxic. Once it enters the environment, it can be transformed into methylmercury with the help of bacteria. Methylmercury can be absorbed through breathing and skin and enriched in fish, leading to food chain transmission and causing serious damage to the human central nervous system, kidneys and immune system.

[0003] Microfluidics is a science and technology characterized by the precise manipulation of fluids in micro-nanoscale space. It has the ability to miniaturize the basic functions of biological and chemical laboratories such as sample preparation, reaction, separation and detection onto a chip of a few square centimeters. Its basic characteristics and greatest advantage are the flexible combination and large-scale integration of multiple unit technologies on an overall controllable micro-platform. It is an interdisciplinary technology involving engineering, physics, chemistry, micromachining and bioengineering. As a new type of biosensor tool, microfluidic chips can be processed to meet the needs of various research fields. Among them, pump-free microfluidic chips can be filled spontaneously, have the advantages of no need for external pumps, easy to carry, customized microfluidic control capabilities and high detection sensitivity, making them play an increasingly important role in the field of on-site trace measurement.

[0004] As an important analytical method, Raman spectroscopy can provide rich information on molecular structure and material composition. With the development of nanotechnology, people have produced sensitive, efficient, repeatable and uniform SERS active substrates through various chemical synthesis and nano-processing methods. Surface enhanced Raman spectroscopy has been widely studied. By enhancing the ordinary Raman scattering signal by up to one million times, it has a sensitivity comparable to that of fluorescence detection methods. In addition to high sensitivity and low detection limit, the SERS method also has the characteristics of "fingerprint spectrum", water stability and high spectral peak resolution, making parallel detection of multiple components possible. Therefore, SERS can be used to detect multiple targets in parallel to obtain rich and diverse spectral information, bringing new opportunities for the detection of various heavy metal ions. Summary of the invention

[0005] The purpose of the present invention is to solve the problems of poor stability and low sensitivity of traditional liquid sol SERS substrates and difficulty in on-site trace inspection of heavy metal ions, and to provide a method for preparing a graded micro-bowl inner cone array SERS substrate and a pump-free microfluidic chip using the substrate.

[0006] To achieve the above purpose, the technical solution provided by the present invention is:

[0007] A preparation method of a hierarchical micro-bowl inner cone array SERS substrate and its application are mainly divided into two parts. The first is to prepare a micro-bowl inner cone array SERS substrate with a micro-nano hierarchical structure, and achieve the improvement of the substrate SERS signal enhancement effect by combining the micrometer scale and nanometer scale structures; then, a pump-free microfluidic chip is prepared according to the prepared micro-bowl inner cone array SERS substrate, and the micro-bowl inner cone array SERS substrate modified with an aptamer is embedded in the detection area of ​​the chip, so as to be used for on-site trace identification and detection of two heavy metal ions, lead and mercury.

[0008] The method for preparing the graded micro-bowl inner cone array SERS substrate comprises the following steps:

[0009] Step S1, 2 μm single-layer PS microspheres were self-assembled on PET;

[0010] The glass slide was treated to be hydrophilic, and deionized water was added to the hydrophilic surface of the glass slide to form a water film. A 5% mass fraction of 2μm PS microsphere mother solution was mixed with ethanol at a volume ratio of 1:1.5, and the prepared PS microsphere ethanol mixture was added drop by drop on the corners of the glass slide. The microspheres self-assembled into a single layer on the water film. After natural air drying, the single layer of PS microspheres was transferred from the glass slide to the surface of deionized water in a beaker. The single layer of PS microspheres was transferred to the PET film by the pulling method. After natural drying, a PET film with a single layer of PS microspheres was obtained.

[0011] Step S2, forming a micro-bowl array by inverting the photoresist;

[0012] The PET film with a single layer of PS microspheres was placed on a 110°C hot plate for 2 min, then the photoresist was poured and imprinted with a polydimethylsiloxane (PDMS) mold for 15 min. After 365 nm UV curing, the cured photoresist was peeled off and immersed in a toluene solution for 10 min to dissolve the PS microspheres and form a microbowl array on the photoresist;

[0013] Step S3, 200 nm monolayer PS nanospheres self-assembled in the bowl;

[0014] After the substrate prepared in step S2 was subjected to hydrophilic treatment, a 200 nm monolayer of PS nanospheres was transferred from the deionized water surface in the beaker to the photoresist microbowl array by the Czochralski method;

[0015] Step S4, ICP etching the nanospheres to form a cone array in the bowl;

[0016] The photoresist substrate with 200 nm PS nanospheres self-assembled in the micro-bowl is placed in the chamber of an inductively coupled plasma etcher for etching to form a periodic nanocone array in the micro-bowl, thereby obtaining a photoresist substrate with a cone array in the bowl;

[0017] Step S5, electron beam evaporation of a gold film;

[0018] The obtained bowl-in-cone array photoresist substrate is placed in the chamber of a vacuum coater, and a layer of gold film is deposited at a rate of 0.04nm / s to 0.05nm / s, eventually forming densely arranged gold nanoparticles with a diameter of 30nm on the nanocones to obtain a graded micro-bowl-in-cone array SERS substrate.

[0019] The preferred technical solution provided by the present invention is:

[0020] In step S1, the hydrophilic treatment is specifically as follows: the glass slide is cut into a square of 2.5 cm × 2.5 cm, and then the cut glass slides are placed in an acetone solution and an anhydrous ethanol solution, respectively, for 45 minutes of ultrasonic cleaning. Then, the glass slide is ultrasonically cleaned in deionized water for 30 minutes. After cleaning, the glass slide is transferred to a "piranha solution" made by mixing concentrated sulfuric acid and hydrogen peroxide and soaked for 6 hours. Subsequently, deionized water is used to clean the residual solution on the surface of the glass slide. The dried glass slide is placed in a plasma cleaner for 3 minutes to obtain a hydrophilic surface.

[0021] The present invention provides another preferred technical solution:

[0022] In step S2, the dimensions of the concave groove of the PDMS mold are: 4 mm×3.5 mm×0.1 mm in length×width×depth.

[0023] The present invention provides a further preferred technical solution:

[0024] The step S3 is specifically as follows:

[0025] 10 μL of 5% 200nm PS nanosphere stock solution was mixed with ethanol in a 1:1 volume ratio, and the prepared PS nanosphere ethanol mixture was slowly dripped into a round petri dish (bottom diameter: 142mm, bottom height: 16mm) filled with deionized water. PS nanospheres self-assembled into a monolayer on the surface of deionized water. 1 μL of 2% surfactant TX-100 was dripped into deionized water, and PS nanospheres formed a dense monolayer on the surface of deionized water.

[0026] The photoresist substrate with the micro-bowl array was placed in a plasma cleaning machine for 1 minute to obtain a hydrophilic surface. The substrate was then inserted into deionized water at a 45° tilt angle, gradually approaching the bottom of the monolayer of PS nanospheres, and then the photoresist substrate was pulled out at a constant speed, and the monolayer of PS nanospheres was transferred from the deionized water surface to the photoresist substrate. After natural air drying, the monolayer of PS nanospheres self-assembled in the photoresist micro-bowl array.

[0027] Furthermore,

[0028] The etching parameters of step S4 are specifically as follows: RF power is 55 W, ICP power is 100 W, process chamber vacuum is 18-20 mTorr, oxygen flow rate is 30 sccm, and etching time is 120 s.

[0029] Preferably,

[0030] The method further comprises step S6, testing the uniformity of the substrate Raman spectrum;

[0031] MGITC was modified on the substrate, and the Raman signals at different positions of the substrate were detected to test the uniformity of the substrate;

[0032] The step S6 is specifically as follows: placing the SERS substrate in a plasma cleaning machine for hydrophilic treatment and then placing it in a -7 The substrate was shaken in a MGITC solution of 100 μM for 2 h, washed with deionized water three times, dried at room temperature, and the uniformity of the prepared substrate was verified by the Raman spectrum obtained by a Raman spectrometer;

[0033] The present invention also provides a technical solution for applying a SERS substrate prepared by a method for preparing a graded micro-bowl inner cone array SERS substrate in a microfluidic chip.

[0034] And, the specific steps include:

[0035] Step S7, substrate functionalization;

[0036] After the SERS substrate is hydrophilic, it is placed in the lead and mercury ion specific aptamer solutions and shaken respectively to fix the aptamers on the substrate, thus completing the aptamer modification on the surface of the SERS substrate;

[0037] Step S8, preparation of a pump-free microfluidic chip and sealing it with a SERS substrate;

[0038] A pump-free microfluidic chip was designed that can embed two SERS substrates and detect lead and mercury ions simultaneously.

[0039] in,

[0040] The aptamer modification method in step S7 is specifically as follows:

[0041] Use a pipette to take 250 μL of 1 μM mercury ion specific aptamer solution and 0.2M NaCl solution into a centrifuge tube, then put the SERS substrate treated by plasma cleaning machine into the centrifuge tube, and put the centrifuge tube into an oscillator for 12 hours. The thiol group at one end of the mercury ion specific aptamer will form an Au-S chemical bond with the gold nanoparticles evenly distributed on the surface of the nanocone to fix the aptamer to the substrate. Rinse with deionized water three times, air dry and wait for embedding into the microfluidic chip.

[0042] Use a pipette to take 250 μL of 1 μM lead ion-specific aptamer solution and 0.2M NaCl solution into a centrifuge tube, then put the SERS substrate treated by plasma cleaning machine into the centrifuge tube, and put the centrifuge tube into an oscillator for 12 hours. The thiol group at one end of the lead ion-specific aptamer will form an Au-S chemical bond with the gold nanoparticles evenly distributed on the surface of the nanocone to fix the aptamer to the substrate. Use a pipette to take 500 μL of 1 μM substrate chain solution into a centrifuge tube, then put the SERS substrate rinsed three times with deionized water into the centrifuge tube, and oscillate for 12 hours. The substrate chain and the lead ion-specific aptamer modified on the SERS substrate combine to form a lead ion-specific double-stranded aptamer. Rinse three times with deionized water, air dry and wait for embedding into the microfluidic chip.

[0043] Preferably,

[0044] The preparation of the pump-free microfluidic chip and the sealing with the SERS substrate in step S8 include the following steps:

[0045] S801, preparation of pump-free microfluidic chip: Design a film plate that can embed two SERS substrates through CAD software. Use a glue spreader to spread 100 micron thick Su8-3035 photoresist all over the silicon wafer, and under the action of 365nm ultraviolet light curing of the ultraviolet photolithography machine, engrave the pattern with channel structure on the film plate on the silicon wafer; put the silicon wafer with pattern into a culture dish. Mix PDMS and curing agent in a ratio of 10:1, remove the bubbles inside, pour into the culture dish, heat and mold to form a microfluidic chip; the length of the chip is 38mm, the width of the chip is about 18mm, the thickness of the chip is about 5mm, the microchannel inside is about 200μm wide and 100um high, and the length of the reserved substrate groove area is 4mm, the width is 3.5mm, and the height is 0.3mm.

[0046] S802, Hydrophilic treatment of pump-free microfluidic chip: In order to improve the self-driving ability of the microfluidic chip, the microfluidic chip was treated with oxygen plasma for 90 seconds and then immediately placed in a culture dish containing PEG heated to 150°C for 25 minutes. After cooling to room temperature, the microfluidic chip was rinsed twice with isopropanol and deionized water to remove residual PEG.

[0047] S803, sealing of pump-free microfluidic chip and SERS substrate: put the PDMS chip and glass sheet into a plasma cleaner, first vacuum treat for 90 seconds, then treat with oxygen plasma for 90 seconds, take out the chip after completion, and use tweezers to embed the SERS substrate incubated with mercury ion-specific aptamer and lead ion-specific double-stranded aptamer into the groove reserved in the PDMS chip, then fit the glass sheet and PDMS chip tightly to complete the sealing assembly of the substrate and the microfluidic chip.

[0048] The present invention also provides a technical solution for the application of a microfluidic chip prepared by a graded micro-bowl inner cone array SERS substrate in the detection of trace amounts of heavy metal ions, the specific steps of which are:

[0049] Step S9, dripping a solution containing lead and mercury ions into the prepared microfluidic chip, statically incubating for 30 minutes, and performing Raman detection;

[0050] 20 μL of solutions containing different concentrations of lead and mercury ions were injected into several prepared pump-free microfluidic chips. Under the combined effect of the surface tension of the channel and the pressure generated by the liquid surface pressure difference at the inlet, the fluid flowed spontaneously in the channel without the need for an external injection pump, and could flow through the entire microfluidic channel within 5 seconds. It was then left to stand for 30 minutes to provide sufficient reaction time for the lead and mercury ions in the solution and the specific aptamers on the SERS substrate. Raman signals were detected by Raman spectrometer.

[0051] The Raman spectrometer parameters are set as follows: the excitation light source is 633 nm, the laser power is 0.25-0.3 mW, the integration time is 1 s, and the number of integrations is 1 time.

[0052] The beneficial effects of the present invention are:

[0053] 1. The bowl-shaped and cone-shaped structures of the graded micro-bowl inner cone array SERS substrate have better light trapping effects, can better enhance the Raman scattering effect, have better detection sensitivity, and the use of the substrate is simple and flexible.

[0054] 2. By embedding the SERS micro-nano substrate into a pump-free microfluidic chip, combined with the characteristics of the pump-free microfluidic chip, such as fast processing speed, low sample consumption, no need for an external injection pump, and the high sensitivity and non-destructive detection of the SERS substrate, trace detection of a small amount of multiple heavy metal ion samples can be performed quickly and efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a schematic diagram of the preparation process of the hierarchical micro-bowl inner cone array SERS substrate of the present invention;

[0056] Figure 2This is a physical picture of the SERS substrate;

[0057] Figure 3 To detect the uniformity of SERS signals of MGITC on the substrate;

[0058] Figure 4 Schematic diagram of lead and mercury ion detection mechanism;

[0059] Figure 5 Physical picture of the pump-free microfluidic chip and schematic diagram of lead and mercury ion detection;

[0060] Figure 6 Concentration gradient Raman spectra of lead and mercury ions and their corresponding linear regression curves. DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings.

[0062] Figure 1 It is a schematic diagram of the preparation process of the graded micro-bowl inner cone array SERS substrate proposed in the present invention.

[0063] The substrate is prepared by the following steps:

[0064] Step S1, 2 μm single-layer PS microspheres were self-assembled on PET;

[0065] The glass slide was treated to be hydrophilic, and deionized water was added to the hydrophilic surface of the glass slide to form a water film. A 5% mass fraction of 2μm PS microsphere mother solution was mixed with ethanol at a volume ratio of 1:1.5, and the prepared PS microsphere ethanol mixture was added drop by drop on the corners of the glass slide. The microspheres self-assembled into a single layer on the water film. After natural air drying, the single layer of PS microspheres was transferred from the glass slide to the surface of deionized water in a beaker. The single layer of PS microspheres was transferred to the PET film by the pulling method. After natural drying, a PET film with a single layer of PS microspheres was obtained.

[0066] Step S2, forming a micro-bowl array by inverting the photoresist;

[0067] The PET film with a single layer of PS microspheres was placed on a 110°C hot plate for 2 min, then the photoresist was poured and imprinted with a polydimethylsiloxane (PDMS) mold for 15 min. After 365 nm UV curing, the cured photoresist was peeled off and immersed in a toluene solution for 10 min to dissolve the PS microspheres and form a microbowl array on the photoresist;

[0068] Step S3, 200 nm monolayer PS nanospheres self-assembled in the bowl;

[0069] After the substrate prepared in step S2 was subjected to hydrophilic treatment, a 200 nm monolayer of PS nanospheres was transferred from the deionized water surface in the beaker to the photoresist microbowl array by the Czochralski method;

[0070] Step S4, ICP etching the nanospheres to form a cone array in the bowl;

[0071] The photoresist substrate with 200nm PS nanospheres self-assembled in the micro-bowl was placed in the chamber of the inductively coupled plasma etcher for etching. The specific etching parameters were: RF power of 55W, ICP power of 100W, process chamber vacuum of 18-20mTorr, oxygen flow rate of 30sccm, and etching time of 120s. A periodic nanocone array was formed in the micro-bowl to obtain a photoresist substrate with a cone array in the bowl;

[0072] Step S5, electron beam evaporation deposits a layer of gold film, the actual picture of the substrate is as follows Figure 2 As shown;

[0073] The obtained bowl-in-cone array photoresist substrate is placed in the chamber of a vacuum coater, and a layer of gold film is deposited at a rate of 0.04nm / s to 0.05nm / s, eventually forming densely arranged gold nanoparticles with a diameter of 30nm on the nanocones to obtain a graded micro-bowl-in-cone array SERS substrate.

[0074] Step S6, substrate Raman spectrum uniformity test, the test result is as follows Figure 3 As shown;

[0075] The SERS substrate was placed in a plasma cleaning machine for hydrophilic treatment and then placed in a -7 The substrate was shaken in MGITC solution for 2 h, rinsed with deionized water three times, and dried at room temperature. The uniformity of the prepared substrate was verified by Raman spectra obtained by Raman spectrometer. The calculated spectrum was 1614 cm -1 The relative standard deviation is 4.6%, and the substrate has good uniformity;

[0076] The specific steps of using the hierarchical micro-bowl inner cone array SERS substrate of the present invention to prepare a microfluidic chip are as follows:

[0077] Step S7, substrate functionalization;

[0078] Use a pipette to take 250 μL of 1 μM mercury ion specific aptamer solution and 0.2M NaCl solution into a centrifuge tube, then put the SERS substrate treated by plasma cleaning machine into the centrifuge tube, and put the centrifuge tube into an oscillator for 12 hours. The thiol group at one end of the mercury ion specific aptamer will form an Au-S chemical bond with the gold nanoparticles evenly distributed on the surface of the nanocone to fix the aptamer to the substrate. Rinse with deionized water three times, air dry and wait for embedding into the microfluidic chip.

[0079] Use a pipette to take 250 μL of 1 μM lead ion-specific aptamer solution and 0.2M NaCl solution into a centrifuge tube, then put the SERS substrate treated by plasma cleaning machine into the centrifuge tube, and put the centrifuge tube into an oscillator for 12 hours. The thiol group at one end of the lead ion-specific aptamer will form an Au-S chemical bond with the gold nanoparticles evenly distributed on the surface of the nanocone to fix the aptamer to the substrate. Use a pipette to take 500 μL of 1 μM substrate chain solution into a centrifuge tube, then put the SERS substrate rinsed three times with deionized water into the centrifuge tube, and oscillate for 12 hours. The substrate chain and the lead ion-specific aptamer modified on the SERS substrate combine to form a lead ion-specific double-stranded aptamer. Rinse three times with deionized water, air dry and wait for embedding into the microfluidic chip.

[0080] Step S8, preparation of pump-free microfluidic chip and sealing with SERS substrate, the actual picture after sealing is as follows Figure 5 As shown; it is divided into the following three steps:

[0081] S801, preparation of pump-free microfluidic chip: Design a film plate that can embed two SERS substrates through CAD software. Use a glue spreader to spread 100 micron thick Su8-3035 photoresist all over the silicon wafer, and under the action of 365nm ultraviolet light curing of the ultraviolet photolithography machine, engrave the pattern with channel structure on the film plate on the silicon wafer; put the silicon wafer with pattern into a culture dish. Mix PDMS and curing agent in a ratio of 10:1, remove the bubbles inside, pour into the culture dish, heat and mold to form a microfluidic chip; the length of the chip is 38mm, the width of the chip is about 18mm, the thickness of the chip is about 5mm, the microchannel inside is about 200μm wide and 100um high, and the length of the reserved substrate groove area is 4mm, the width is 3.5mm, and the height is 0.3mm.

[0082] S802, Hydrophilic treatment of pump-free microfluidic chip: In order to improve the self-driving ability of the microfluidic chip, the microfluidic chip was treated with oxygen plasma for 90 seconds and then immediately placed in a culture dish containing PEG heated to 150°C for 25 minutes. After cooling to room temperature, the microfluidic chip was rinsed twice with isopropanol and deionized water to remove residual PEG.

[0083] S803, sealing of pump-free microfluidic chip and SERS substrate: put the PDMS chip and glass sheet into a plasma cleaner, first vacuum treat for 90 seconds, then treat with oxygen plasma for 90 seconds, take out the chip after completion, and use tweezers to embed the SERS substrate incubated with mercury ion-specific aptamer and lead ion-specific double-stranded aptamer into the groove reserved in the PDMS chip, then fit the glass sheet and PDMS chip tightly to complete the sealing assembly of the substrate and the microfluidic chip.

[0084] The specific method for using the prepared microfluidic chip for heavy metal ion trace detection is as follows:

[0085] Step S9, heavy metal ion trace detection, the SERS detection principle of lead and mercury ions is as follows Figure 4 As shown;

[0086] 20 μL of solutions containing different concentrations of lead and mercury ions were injected into several prepared pump-free microfluidic chips. Under the combined effect of the surface tension of the channel and the pressure generated by the liquid surface pressure difference at the inlet, the fluid flows spontaneously in the channel without the need for an external injection pump, and can flow through the entire microfluidic channel within 5 seconds. It was then left to stand for 30 minutes to provide sufficient reaction time for the lead and mercury ions in the solution and the specific aptamers on the SERS substrate. The Raman signal was detected by a Raman spectrometer. The concentration gradient Raman spectra of lead and mercury ions and their corresponding linear regression curves are shown in Figure 2. Figure 6 As shown in the figure, it can be seen that the detection limit of lead and mercury ions of the present invention can reach 10 -12 M.

[0087] In this embodiment, the parameters of the Raman spectrometer are set as follows: the excitation light source is 633 nm, the laser power is 0.25-0.3 mW, the integration time is 1 s, and the number of integrations is 1 time.

[0088] 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 hierarchical micro-bowl inner cone array SERS substrate, characterized in that: The following steps are involved: Step S1, self-assembly of a single layer of PS microspheres on PET; The glass slide is subjected to hydrophilic treatment, and deionized water is added to the hydrophilic surface of the glass slide to form a water film; The PS microsphere stock solution was mixed with ethanol, and the prepared PS microsphere ethanol mixture was added drop by drop on the edge of the glass slide, and the microspheres self-assembled into a single layer on the water film; After natural air drying, the monolayer of PS microspheres was transferred from the glass slide to the surface of deionized water in a beaker; The monolayer PS microspheres were transferred onto the PET film by the Czochralski method; After natural drying, a PET film with a single layer of PS microspheres was obtained; Step S2, forming a micro-bowl array by inverting the photoresist; The PET film with a single layer of PS microspheres obtained in step S1 is placed on a heating plate for heating, and then a photoresist is poured and imprinted with a polydimethylsiloxane (PDMS) mold; after UV curing, the cured photoresist is peeled off and immersed in a toluene solution, and after the PS microspheres are dissolved, a microbowl array is formed on the photoresist; Step S3, a single layer of PS nanospheres is self-assembled in the microbowl; After the substrate prepared in step S2 is subjected to hydrophilic treatment, a single layer of PS nanospheres is transferred from the deionized water surface to the photoresist microbowl array by a Czochralski method; Step S4, ICP etching the nanospheres to form a cone array in the bowl; Placing the photoresist substrate with PS nanospheres self-assembled in the micro-bowl obtained in step S3 in the chamber of an inductively coupled plasma etcher for etching to form a periodic nanocone array in the micro-bowl, thereby obtaining a photoresist substrate with a cone array in the bowl; Step S5, electron beam evaporation of a gold film; The bowl-in-cone array photoresist substrate obtained in step S4 is placed in the chamber of a vacuum coater, a layer of gold film is deposited, and finally gold nanoparticles are formed on the nanocones to obtain a graded micro-bowl-in-cone array SERS substrate, thereby completing the preparation of the graded micro-bowl-in-cone array SERS substrate.

2. The method for preparing a hierarchical micro-bowl inner cone array SERS substrate according to claim 1, characterized in that: In the step S1, the hydrophilic treatment is specifically as follows: The slides were cut into 2.5 cm × 2.5 cm squares, and then the cut slides were placed in acetone solution and anhydrous ethanol solution for 45 minutes of ultrasonic cleaning; The slides were ultrasonically cleaned in deionized water for 30 min; Transfer the slides to a "piranha solution" made of a mixture of concentrated sulfuric acid and hydrogen peroxide and soak for 6 hours; Use deionized water to clean the residual solution on the surface of the slide; The dried glass slide was placed in a plasma cleaning machine for 3 minutes to obtain a hydrophilic surface.

3. The method for preparing a hierarchical micro-bowl inner cone array SERS substrate according to claim 1, characterized in that: The step S3 is specifically as follows: 10 μL of 5% 200 nm PS nanosphere stock solution was mixed with ethanol in a 1:1 volume ratio, and the prepared PS nanosphere ethanol mixture was slowly dripped drop by drop into a round culture dish filled with deionized water. The PS nanospheres self-assembled into a monolayer on the surface of the deionized water. When 1 μL of 2% surfactant TX-100 was dropped into deionized water, PS nanospheres formed a dense monolayer on the surface of deionized water; The photoresist substrate with the micro-bowl array was placed in a plasma cleaning machine for 1 minute to obtain a hydrophilic surface; The substrate was inserted into deionized water at an angle of 45°, gradually approaching the bottom of the monolayer of PS nanospheres, and then the photoresist substrate was pulled out at a constant speed, and the monolayer of PS nanospheres was transferred from the deionized water surface to the photoresist substrate; After natural air drying, a single layer of PS nanospheres self-assembled in the photoresist microbowl array.

4. The method for preparing a hierarchical micro-bowl inner cone array SERS substrate according to claim 1, characterized in that: The etching parameters of step S4 are specifically as follows: RF power is 55 W, ICP power is 100 W, process chamber vacuum is 18-20 mTorr, oxygen flow rate is 30 sccm, and etching time is 120 s.

5. The method for preparing a hierarchical micro-bowl inner cone array SERS substrate according to claim 1, characterized in that: The method further comprises step S6, testing the uniformity of the substrate Raman spectrum; MGITC was modified on the substrate, and the Raman signals at different positions of the substrate were detected to test the uniformity of the substrate; Specifically, the SERS substrate was placed in a plasma cleaning machine for hydrophilic treatment and then placed in a -7 The prepared substrate was shaken in a MGITC solution of 100 M for 2 h, taken out and rinsed with deionized water for three times, and dried at room temperature. The uniformity of the prepared substrate was verified by the Raman spectrum obtained by a Raman spectrometer.

6. Application of a SERS substrate prepared by the method for preparing a graded micro-bowl inner cone array SERS substrate as claimed in any one of claims 1 to 5 in a microfluidic chip.

7. The use of the hierarchical micro-bowl inner cone array SERS substrate in a microfluidic chip according to claim 6, characterized in that: The specific steps are: Step S7, substrate functionalization; After the SERS substrate is hydrophilic, it is placed in the lead and mercury ion specific aptamer solutions and shaken respectively to fix the aptamers on the substrate, thus completing the aptamer modification on the surface of the SERS substrate; Step S8, preparation of a pump-free microfluidic chip and sealing it with a SERS substrate; A pump-free microfluidic chip was designed that can embed two SERS substrates and detect lead and mercury ions simultaneously.

8. The use of the hierarchical micro-bowl inner cone array SERS substrate in a microfluidic chip according to claim 7, characterized in that: The aptamer modification method in step S7 is specifically as follows: Use a pipette to take 250 μL of 1 μM mercury ion-specific aptamer solution and 0.2 M NaCl solution into a centrifuge tube, then put the SERS substrate treated by a plasma cleaner into the centrifuge tube, and put the centrifuge tube into an oscillator for 12 hours. The thiol group at one end of the mercury ion-specific aptamer will form an Au-S chemical bond with the gold nanoparticles evenly distributed on the surface of the nanocone to fix the aptamer to the substrate. Rinse with deionized water three times and air-dry before embedding into the microfluidic chip; Use a pipette to take 250 μL of 1 μM lead ion-specific aptamer solution and 0.2 M NaCl solution and put them into a centrifuge tube. Then put the SERS substrate treated by a plasma cleaner into the centrifuge tube. Put the centrifuge tube into an oscillator and oscillate it for 12 hours. The thiol group at one end of the lead ion-specific aptamer will form an Au-S chemical bond with the gold nanoparticles evenly distributed on the surface of the nanocone to fix the aptamer to the substrate. Use a pipette to take 500 μL of a 1 μM substrate chain solution and put it into a centrifuge tube. Then, after washing the SERS substrate three times with deionized water, put it into the centrifuge tube and shake it for 12 hours. The substrate chain and the lead ion-specific aptamer modified on the SERS substrate will combine to form a lead ion-specific double-stranded aptamer. Rinse with deionized water three times and air-dry before embedding into the microfluidic chip.

9. The use of the hierarchical micro-bowl inner cone array SERS substrate in a microfluidic chip according to claim 7, characterized in that: The step S8 specifically includes the following steps: S801, Preparation of pump-free microfluidic chip: Design a film plate that can embed two SERS substrates using CAD software; A 100-micron-thick Su8-3035 photoresist is spread over the entire silicon wafer by a coating machine, and the pattern with a channel structure on the film plate is engraved on the silicon wafer under the action of 365nm ultraviolet light curing of the ultraviolet photolithography machine; Place the patterned silicon wafer into a petri dish; PDMS and curing agent were mixed in a ratio of 10:1, and after removing the bubbles inside, they were poured into a culture dish and heated to form a microfluidic chip. S802, Hydrophilic treatment of pump-free microfluidic chip: In order to improve the self-driving ability of the microfluidic chip, the microfluidic chip was treated with oxygen plasma for 90 seconds and then immediately placed in a culture dish containing PEG heated to 150°C for 25 minutes. After cooling to room temperature, the microfluidic chip was rinsed twice with isopropanol and deionized water to remove residual PEG; S803, sealing of pump-free microfluidic chip and SERS substrate: The PDMS chip and glass slide were placed in a plasma cleaner, first vacuum treated for 90 seconds, and then treated with oxygen plasma for 90 seconds. After completion, the chip was taken out and tweezers were used to embed the SERS substrates incubated with mercury ion-specific aptamers and lead ion-specific double-stranded aptamers into the grooves reserved for the PDMS chip. The glass slide was then tightly fitted to the PDMS chip to complete the sealing assembly of the substrate and the microfluidic chip.

10. An application of a microfluidic chip prepared by a graded micro-bowl inner cone array SERS substrate as claimed in any one of claims 6 to 9, characterized in that: The method comprises step S9, detecting trace amounts of heavy metal ions; A solution containing lead and mercury ions was dripped into the prepared microfluidic chip, incubated statically for 30 min, and Raman detection was performed; 20 μL of lead and mercury ion solutions containing different concentrations were injected into multiple prepared pump-free microfluidic chips; Under the combined effect of the surface tension of the channel and the pressure generated by the liquid surface pressure difference at the inlet, the fluid flows spontaneously in the channel without the need for an external syringe pump and flows through the entire microfluidic channel within 5 seconds; The mixture was allowed to stand for 30 min to provide sufficient reaction time for the lead and mercury ions in the solution and the specific aptamers on the SERS substrate; The Raman signal is detected by a Raman spectrometer.

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