Preparation method and application of a graded micro-bowl inner cone array SERS substrate

Abstract

The present invention discloses a preparation method and application of a graded micro-bowl inner cone array SERS substrate, which belongs to the field of surface enhanced Raman spectroscopy detection and microfluidics technology. The preparation steps include: S1, self-assembly of a single layer of PS microspheres on PET; S2, photoresist molding to form a micro-bowl array; S3, self-assembly of a single layer of PS nanospheres in the bowl; S4, ICP etching of nanospheres to form a bowl inner cone array; S5, electron beam evaporation of a layer of gold film; S6, substrate characterization; S7, substrate functionalization; S8, preparation of a microfluidic chip and sealing with the SERS substrate; S9, trace detection of heavy metal ions. The graded micro-bowl inner cone array SERS substrate proposed by the present invention is stable in nature, not easily oxidized, has a uniform structure, has a large specific surface area, contains abundant hot spots, and its micro-nano graded structure can better regulate the incident electromagnetic light field, thereby enhancing the Raman scattering effect, which is of great significance for the trace detection of the object to be detected.

Description

Technical Field

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

[0002] Heavy metal pollution can accumulate in organisms and spread through the food chain, reducing the diversity and abundance of aquatic species and contributing to the degradation of aquatic ecosystems. Their non-biodegradability, persistence, and biomagnification pose a serious threat to human health. According to the World Health Organization's tolerable daily intake (TIA) for various heavy metals, the TIA for mercury and lead is less than 1 μg / kg / day. Heavy metal mercury, in particular, is extremely toxic. Once it enters the environment, it can be transformed into methylmercury by bacteria. Methylmercury can be absorbed through respiration and skin, and accumulates in fish, leading to its spread through the food chain and causing serious damage to the human central nervous system, kidneys, and immune system.

[0003] Microfluidics is a scientific technology characterized by the precise manipulation of fluids at the micro- and nanoscale. It has the ability to miniaturize basic laboratory functions in biology and chemistry, such as sample preparation, reaction, separation, and detection, onto a chip of a few square centimeters. Its basic feature and greatest advantage is 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 not requiring an external pump, are easy to carry, have customized microfluidic control capabilities, and have 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 a wealth of information on molecular structure and material composition. With the development of nanotechnology, people have created sensitive, efficient, repeatable, and uniform SERS-active substrates through various chemical synthesis and nanofabrication methods. Surface-enhanced Raman spectroscopy has been widely studied. By enhancing ordinary Raman scattering signals by up to a million times, it has a sensitivity comparable to that of fluorescence detection methods. In addition to high sensitivity and low detection limits, the SERS method also has the characteristics of "fingerprinting", water stability, and high spectral peak resolution, which makes the 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 detection 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 this substrate.

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

[0007] A preparation method and application of a hierarchical micro-bowl inner cone array SERS substrate 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 an improved SERS signal enhancement effect of the substrate by combining micron-scale and nano-scale structures; then, a pump-free microfluidic chip is prepared based on 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, thereby being used for on-site trace identification and detection of two heavy metal ions, lead and mercury.

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

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

[0010] A glass slide was treated to make it hydrophilic, and deionized water was added to the hydrophilic surface to form a water film. A 5% mass fraction of a 2μm PS microsphere stock solution was mixed with ethanol at a volume ratio of 1:1.5. This PS microsphere-ethanol mixture was then added dropwise to the corners of the glass slide, where the microspheres self-assembled into a monolayer on the water film. After air-drying, the monolayer of PS microspheres was transferred from the glass slide to the surface of deionized water in a beaker. The monolayer of PS microspheres was then transferred to a PET film using the Czochralski method. After air-drying, a PET film with a monolayer of PS microspheres was obtained.

[0011] Step S2, forming a micro-bowl array by performing photoresist molding;

[0012] A PET film with a single layer of PS microspheres was placed on a 110°C hotplate for 2 minutes, followed by pouring photoresist and imprinting with a polydimethylsiloxane (PDMS) mold for 15 minutes. After curing with 365nm UV light, the cured photoresist was stripped and immersed in a toluene solution for 10 minutes 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 hydrophilized, 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-in-bowl array;

[0016] The photoresist substrate with 200 nm PS nanospheres self-assembled in the micro-bowl was 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 an in-bowl cone array.

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

[0018] The obtained bowl-in-cone array photoresist substrate was placed in the chamber of a vacuum coater, and a layer of gold film was deposited at a rate of 0.04nm / s to 0.05nm / s. Finally, densely arranged gold nanoparticles with a diameter of 30nm were formed 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 2.5 cm × 2.5 cm squares, and then the cut glass slides are placed in acetone solution and anhydrous ethanol solution respectively, and ultrasonically cleaned for 45 minutes. 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, the residual solution on the surface of the glass slide is washed with deionized water. 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: length×width×depth: 4 mm×3.5 mm×0.1 mm.

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

[0024] The step S3 is specifically as follows:

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

[0026] The photoresist substrate with the micro-bowl array was placed in a plasma cleaner for one minute to create a hydrophilic surface. The substrate was then inserted into deionized water at a 45° angle, gradually approaching the bottom of the monolayer of PS nanospheres. The photoresist substrate was then pulled out at a constant speed, transferring the monolayer of PS nanospheres from the deionized water surface to the photoresist substrate. After air-drying, the monolayer of PS nanospheres self-assembled within the photoresist micro-bowl array.

[0027] Further,

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

[0029] Preferably,

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

[0031] MGITC was modified on the substrate, and the Raman signals at different positions on 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 MGITC solution of 400 M for 2 h, washed 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.

[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 to fix the aptamers on the substrate, thus completing the aptamer modification on the SERS substrate surface;

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

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

[0039] in,

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

[0041] Use a pipette to place 250 μL of a 1 μM mercury ion-specific aptamer solution and a 0.2 M NaCl solution into a centrifuge tube. Then, place the SERS substrate, which has been hydrophilized using a plasma cleaner, into the centrifuge tube. Oscillate the tube on an oscillator for 12 hours. The thiol group on one end of the mercury ion-specific aptamer forms an Au-S chemical bond with the gold nanoparticles evenly distributed on the nanocone surface, securing the aptamer to the substrate. Rinse three times with deionized water and air-dry before embedding into a microfluidic chip.

[0042] Using a pipette, 250 μL of a 1 μM solution of lead-specific aptamers and a 0.2 M NaCl solution were placed into a centrifuge tube. The SERS substrate, hydrophilized by plasma cleaning, was then placed into the tube and shaken for 12 hours. The thiol groups on one end of the lead-specific aptamers formed Au-S chemical bonds with the gold nanoparticles evenly distributed on the nanocone surfaces, anchoring the aptamers to the substrate. Using a pipette, 500 μL of a 1 μM solution of substrate chains was placed into a centrifuge tube. The SERS substrate, rinsed three times with deionized water, was then placed into the tube and shaken for 12 hours. The substrate chains bound to the lead-specific aptamers modified on the SERS substrate, forming a double-stranded lead-specific aptamer. The tube was rinsed three times with deionized water and air-dried before embedding into a microfluidic chip.

[0043] Preferably,

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

[0045] S801, Preparation of a Pump-Free Microfluidic Chip: CAD software was used to design a film plate capable of embedding two SERS substrates. A 100-micron-thick layer of Su8-3035 photoresist was applied to the entire silicon wafer using a spreader. Using a 365nm UV curing machine, the channel pattern on the film plate was imprinted onto the silicon wafer. The patterned silicon wafer was placed in a Petri dish. PDMS and curing agent were mixed in a 10:1 ratio, and after removing any bubbles, the mixture was poured into a Petri dish and heated to form a molded microfluidic chip. The chip was 38 mm long, 18 mm wide, and 5 mm thick. The microchannels within it were approximately 200 μm wide and 100 μm high. The reserved substrate recess was 4 mm long, 3.5 mm wide, and 0.3 mm high.

[0046] S802, Hydrophilic Treatment of the Pump-Free Microfluidic Chip: To enhance the self-propulsion capability of the microfluidic chip, the chip was treated with oxygen plasma for 90 seconds and then immediately placed in a Petri dish containing PEG heated to 150°C for 25 minutes. After cooling to room temperature, the chip was rinsed twice with isopropyl alcohol and deionized water to remove any residual PEG.

[0047] S803, sealing the pump-free microfluidic chip and the SERS substrate: Place the PDMS chip and glass slide in a plasma cleaner, first vacuum treat for 90 seconds, and then treat with oxygen plasma for 90 seconds. After completion, take out the chip and use tweezers to embed the SERS substrate incubated with mercury ion-specific aptamers and lead ion-specific double-stranded aptamers into the grooves reserved in the PDMS chip. Then, tightly fit the glass slide and the PDMS chip 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 with a graded micro-bowl inner cone array SERS substrate in the detection of trace amounts of heavy metal ions, which specifically comprises the following steps:

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

[0050] 20 μL of solutions containing different concentrations of lead and mercury ions were injected into several pre-prepared pump-free microfluidic chips. The fluids spontaneously flowed within the channels, driven by the combined effects of surface tension and the pressure differential at the inlet. Without the need for an external syringe pump, the fluids flowed through the entire microfluidic channel within 5 seconds. The solution was then allowed to stand for 30 minutes to allow sufficient time for the lead and mercury ions in the solution to react with the specific aptamers on the SERS substrate. Raman signals were detected using a Raman spectrometer.

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

[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 effect, can better enhance the Raman scattering effect, have better detection sensitivity, and the substrate is simple and flexible to use.

[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 syringe pump, and the high sensitivity and non-destructive detection of the SERS substrate, trace detection of small amounts of multiple heavy metal ion samples can be performed quickly and efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 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 Detection of the uniformity of SERS signals of MGITC on the substrate;

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

[0059] Figure 5 A physical image of the pump-free microfluidic chip and a schematic diagram of lead and mercury ion detection;

[0060] Figure 6 Raman spectra of lead and mercury ion concentration gradients 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 with reference to the accompanying drawings.

[0062] Figure 1 This 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 monolayer PS microspheres were self-assembled on PET;

[0065] A glass slide was treated to make it hydrophilic, and deionized water was added to the hydrophilic surface to form a water film. A 5% mass fraction of a 2μm PS microsphere stock solution was mixed with ethanol at a volume ratio of 1:1.5. This PS microsphere-ethanol mixture was then added dropwise to the corners of the glass slide, where the microspheres self-assembled into a monolayer on the water film. After air-drying, the monolayer of PS microspheres was transferred from the glass slide to the surface of deionized water in a beaker. The monolayer of PS microspheres was then transferred to a PET film using the Czochralski method. After air-drying, a PET film with a monolayer of PS microspheres was obtained.

[0066] Step S2, forming a micro-bowl array by performing photoresist molding;

[0067] A PET film with a single layer of PS microspheres was placed on a 110°C hotplate for 2 minutes, followed by pouring photoresist and imprinting with a polydimethylsiloxane (PDMS) mold for 15 minutes. After curing with 365nm UV light, the cured photoresist was stripped and immersed in a toluene solution for 10 minutes 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 hydrophilized, 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-in-bowl array;

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

[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 was placed in the chamber of a vacuum coater, and a layer of gold film was deposited at a rate of 0.04nm / s to 0.05nm / s. Finally, densely arranged gold nanoparticles with a diameter of 30nm were formed on the nanocones to obtain a graded micro-bowl-in-cone array SERS substrate.

[0074] Step S6: substrate Raman spectrum uniformity test, the test results are 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, washed 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 place 250 μL of a 1 μM mercury ion-specific aptamer solution and a 0.2 M NaCl solution into a centrifuge tube. Then, place the SERS substrate, which has been hydrophilized using a plasma cleaner, into the centrifuge tube. Oscillate the tube on an oscillator for 12 hours. The thiol group on one end of the mercury ion-specific aptamer forms an Au-S chemical bond with the gold nanoparticles evenly distributed on the nanocone surface, securing the aptamer to the substrate. Rinse three times with deionized water and air-dry before embedding into a microfluidic chip.

[0079] Using a pipette, 250 μL of a 1 μM solution of lead-specific aptamers and a 0.2 M NaCl solution were placed into a centrifuge tube. The SERS substrate, hydrophilized by plasma cleaning, was then placed into the tube and shaken for 12 hours. The thiol groups on one end of the lead-specific aptamers formed Au-S chemical bonds with the gold nanoparticles evenly distributed on the nanocone surfaces, anchoring the aptamers to the substrate. Using a pipette, 500 μL of a 1 μM solution of substrate chains was placed into a centrifuge tube. The SERS substrate, rinsed three times with deionized water, was then placed into the tube and shaken for 12 hours. The substrate chains bound to the lead-specific aptamers modified on the SERS substrate, forming a double-stranded lead-specific aptamer. The tube was rinsed three times with deionized water and air-dried before embedding into a 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 a Pump-Free Microfluidic Chip: CAD software was used to design a film plate capable of embedding two SERS substrates. A 100-micron-thick layer of Su8-3035 photoresist was applied to the entire silicon wafer using a spreader. Using a 365nm UV curing machine, the channel pattern on the film plate was imprinted onto the silicon wafer. The patterned silicon wafer was placed in a Petri dish. PDMS and curing agent were mixed in a 10:1 ratio, and after removing any bubbles, the mixture was poured into a Petri dish and heated to form a molded microfluidic chip. The chip was 38 mm long, 18 mm wide, and 5 mm thick. The microchannels within it were approximately 200 μm wide and 100 μm high. The reserved substrate recess was 4 mm long, 3.5 mm wide, and 0.3 mm high.

[0082] S802, Hydrophilic Treatment of the Pump-Free Microfluidic Chip: To enhance the self-propulsion capability of the microfluidic chip, the chip was treated with oxygen plasma for 90 seconds and then immediately placed in a Petri dish containing PEG heated to 150°C for 25 minutes. After cooling to room temperature, the chip was rinsed twice with isopropyl alcohol and deionized water to remove any residual PEG.

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

[0084] The specific method for using the prepared microfluidic chip for trace detection of heavy metal ions 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 lead and mercury ions of different concentrations were injected into several prepared pump-free microfluidic chips. Under the combined action 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 allowed 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, and 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.

[0088] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection 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 treated to be hydrophilic, 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. The microspheres self-assembled into a monolayer 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; A single layer of PS microspheres was transferred onto a PET film via 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 performing photoresist molding; The PET film with a single layer of PS microspheres obtained in step S1 is placed on a hot 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 soaked in a toluene solution to dissolve the PS microspheres, forming a microbowl array on the photoresist; Step S3, a single layer of PS nanospheres self-assembles in the microbowl; After the substrate prepared in step S2 is hydrophilized, a monolayer of PS nanospheres is transferred from the deionized water surface to the photoresist microbowl array by the Czochralski method; Step S4, ICP etching the nanospheres to form a cone-in-bowl array; 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-inner-cone array photoresist substrate obtained in step S4 is placed in the chamber of a vacuum coating apparatus, and a layer of gold film is deposited to eventually form gold nanoparticles on the nanocones to obtain a graded micro-bowl-inner-cone array SERS substrate, thereby completing the preparation of the graded micro-bowl-inner-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 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 respectively 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 slide surface; 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 a 5% mass fraction 200 nm PS nanosphere stock solution was mixed with ethanol at a 1:1 volume ratio. The prepared PS nanosphere-ethanol mixture was slowly dripped dropwise into a round Petri 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 cleaner for 1 minute to obtain a hydrophilic surface; The substrate was 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, 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 in step S4 are specifically as follows: RF power of 55 W, ICP power of 100 W, process chamber vacuum of 18-20 mTorr, oxygen flow rate of 30 sccm, and etching time of 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 includes step S6, testing the uniformity of the substrate Raman spectrum; MGITC was modified on the substrate, and the Raman signals at different positions on the substrate were detected to test the uniformity of the substrate. Specifically: Place the SERS substrate in a plasma cleaning machine for hydrophilic treatment and then place it in a -7 The substrate was shaken in a MGITC solution of 100 μM for 2 h, taken out and rinsed with deionized water 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. Use of a SERS substrate prepared by the method for preparing a graded micro-bowl inner cone array SERS substrate according to any one of claims 1 to 5 in a microfluidic chip.

7. Application 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 to fix the aptamers on the substrate, thus completing the aptamer modification on the SERS substrate surface; Step S8, preparation of the pump-free microfluidic chip and sealing it with the SERS substrate; A pump-free microfluidic chip was designed that can embed two SERS substrates and simultaneously detect lead and mercury ions.

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 a 1 μM mercury ion-specific aptamer solution and a 0.2 M NaCl solution into a centrifuge tube. Then, place the SERS substrate, which has been hydrophilically treated by a plasma cleaner, into the centrifuge tube. The centrifuge tube is placed in an oscillator and shaken 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 nanocones, fixing 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 a 1 μM lead ion-specific aptamer solution and a 0.2 M NaCl solution and place it into a centrifuge tube. Then, place the SERS substrate, which has been hydrophilically treated by a plasma cleaner, into the centrifuge tube. The centrifuge tube is then placed in an oscillator and shaken 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 nanocones, fixing the aptamer to the substrate. Use a pipette to take 500 μL of a 1 μM substrate chain solution and place it in a centrifuge tube. Then, after rinsing the SERS substrate three times with deionized water, place it in the centrifuge tube and shake it for 12 hours. The substrate chain binds to the lead ion-specific aptamer modified on the SERS substrate 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. Application 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 be embedded with two SERS substrates using CAD software; A 100-micron-thick Su8-3035 photoresist is spread over the entire silicon wafer using a spreader. The channel pattern on the film is then imprinted onto the silicon wafer using a 365nm UV curing 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 mold to form a microfluidic chip. S802, hydrophilic treatment of pump-free microfluidic chips: 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 that had been 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 the pump-free microfluidic chip to the SERS substrate: The PDMS chip and glass slide were placed in a plasma cleaner, first vacuumed 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 in 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 using 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 minutes, and then 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 effects of the channel surface tension and the pressure difference at the inlet, the fluid flows spontaneously in the channel without the need for an external syringe pump, flowing through the entire microfluidic channel within 5 seconds. The solution 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 was detected by a Raman spectrometer.

Images