Preparation method and application of hierarchical frustum porous light-trapping synergistic SERS substrate

Abstract

This invention discloses a method for preparing a hierarchical frustum-shaped porous synergistic light-trapping SERS substrate and its application, belonging to the field of surface-enhanced Raman spectroscopy and microfluidics. The preparation steps include: S1, self-assembly of monolayer PS microspheres onto a PI film; S2, formation of a frustum structure on the PI surface via ICP etching; S3, removal of residual PS microsphere template by toluene dissolution and formation of a micron-scale pit array using PDMS molding; S4, self-assembly of monolayer PS nanospheres within the micron-scale pits and covering the nanosphere array with SU-8 photoresist; S5, cross-linking of SU-8 with UV exposure and removal of the nanospheres to form nanopores; S6, electron beam evaporation of a gold film; S7, substrate characterization; S8, substrate functionalization; S9, fabrication of a microfluidic chip and sealing with the SERS substrate; S10, heavy metal ion trace detection. The hierarchical frustum-shaped porous synergistic light-trapping SERS substrate proposed in this invention exhibits excellent structural uniformity and chemical stability, good oxidation resistance, and high-density hotspots. Its micro-nano synergistic structure can effectively improve light energy utilization efficiency and form abundant surface plasmon resonance regions, thereby enhancing Raman scattering signals, which has important application value for trace detection of analytes.

Description

Technical Field

[0001] This invention belongs to the field of surface-enhanced Raman spectroscopy detection and microfluidics technology. Specifically, this invention relates to a method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate and its application. Background Technology

[0002] With the continuous development of global industrial manufacturing and the heavy metal economy, the excessive emission of heavy metal ions has become a serious environmental problem. This is especially true for lead ions (Pb). 2+ ) and hexavalent chromium ions (Cr 6+ Lead ions are highly toxic, persistent, and bioaccumulate easily in marine ecosystems and are amplified through the food chain, ultimately damaging the human central nervous system, kidneys, and immune system. Seawater quality standards stipulate that the limit for lead ions in Class I seawater of marine fishing areas should be less than 1 ppb, and the limit for hexavalent chromium ions should be less than 5 ppb.

[0003] Surface-enhanced Raman scattering (SERS) is a mature analytical technique widely used for trace detection of hazardous substances due to its high sensitivity, unique molecular vibrational fingerprint characteristics, and non-destructive testing. The significant enhancement of the SERS signal is mainly attributed to the localized surface plasmon resonance (LSPR) effect induced by noble metal (such as gold, silver, and copper) nanoparticles or nanostructures. LSPR can locally excite strong electromagnetic fields (hot spots) on the metal surface. When the target molecule is located in the hot spot region, its Raman scattering signal intensity is greatly enhanced, with the enhancement factor for a single molecule reaching 10-1. 14 Designing novel SERS substrates with high-density and spatially uniform hotspot regions is helpful in meeting the needs of molecular trace detection and promoting the practical application of SERS. In recent years, spatially extendable three-dimensional (3D) SERS substrates have become a new research hotspot. Compared with traditional two-dimensional planar substrates, three-dimensional structures, through extension along the Z-axis, form a three-dimensional hotspot distribution in three-dimensional space, thereby achieving a stronger Raman signal enhancement effect.

[0004] Microfluidic chips are a cutting-edge micro-nano technology characterized by precise fluid manipulation within a micrometer-scale space. They integrate and miniaturize multiple functions of biological and chemical laboratories onto a chip of just a few square centimeters, hence the name "lab-on-a-chip." Microfluidic chips offer advantages such as small sample volume, low cost, and fast reaction speed, showing broad application prospects in life sciences, chemical analysis, drug screening, and environmental monitoring. In particular, the combination of microfluidic technology and SERS enables highly sensitive detection of trace substances, and is considered one of the most promising detection methods for the future. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of uncontrolled aggregation or sedimentation of colloidal particles in noble metal sol SERS substrates in high salinity or complex environments, and to provide a method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate and a pump-free microfluidic chip using this substrate.

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

[0007] A method for fabricating a hierarchical frustum-shaped porous SERS substrate and its application are disclosed. The overall research process can be divided into two parts. First, a frustum-shaped nanopore array SERS substrate with a micro-nano hierarchical structure is constructed. By introducing nanoscale pores into the frustum structure at the micrometer scale, synergistic coupling of multi-scale structures is achieved, which significantly enhances the local electromagnetic field distribution and SERS signal intensity. Then, the fabricated frustum-shaped nanopore array SERS substrate is integrated into a pump-free microfluidic chip. The SERS substrate modified with specific recognition molecules is embedded in the detection area of ​​the chip to realize the recognition and detection of lead-chromium ions.

[0008] The method for preparing the hierarchical frustum porous synergistic light-trapping SERS substrate includes the following steps:

[0009] Step S1: Monolayer PS microspheres self-assemble on PI film;

[0010] The PI membrane was subjected to hydrophilic treatment using a plasma cleaner. A stock solution of 5% PS microspheres with a particle size of 6 μm was mixed with anhydrous ethanol at a volume ratio of 3:1. The prepared mixture was then added dropwise vertically to a glass petri dish containing deionized water, where the microspheres formed a monolayer membrane structure on the deionized water surface. The monolayer PS microspheres were then transferred from the water surface to the PI membrane surface using a dip-coating method. After natural drying, a PI membrane with a monolayer PS microspheres was obtained.

[0011] Step S2: Form a frustum structure on the PI surface based on ICP etching;

[0012] A PI film with a single layer of PS microspheres is placed in the reaction chamber of an inductively coupled plasma etching machine for oxygen plasma etching. The PS microspheres are used as a mask to form a periodic frustum array on the surface of the PI film.

[0013] Step S3: Toluene is used to dissolve and remove the residual PS microsphere template, and a micron-scale pit array is formed by molding with PDMS.

[0014] The etched PI film was placed in toluene solution for 10 minutes to dissolve the residual PS microspheres. PDMS was poured onto the surface of the PI film and placed on a 70°C heating plate for 2 hours. After the PDMS was completely cured, it was peeled off from the PI film to obtain a PDMS pit array.

[0015] Step S4: Monolayer PS nanospheres are self-assembled in micron-sized pits and SU-8 photoresist is dropped onto the nanosphere array to cover it;

[0016] After hydrophilic treatment, PDMS with pits is transferred into the pits by a Czochralski method, and SU-8 photolithography is applied to the PS nanospheres and the surface of the PDMS pits.

[0017] Step S5: Crosslink SU-8 with ultraviolet light and remove nanospheres to form nanopores;

[0018] After being exposed and cured by 365 nm ultraviolet light, the SU-8 photoresist was peeled off from the PDMS, and the cured photoresist was immersed in toluene solution for 10 minutes to remove the PS nanospheres and form a nanoporous structure on the surface of the frustum.

[0019] Step S6: Electron beam evaporation deposits a gold film;

[0020] The prepared frustum nanopore array photoresist substrate was placed in the chamber of a vacuum deposition system, and a 30 nm gold film was uniformly deposited under high vacuum conditions at a deposition rate of 0.02 nm / s to 0.03 nm / s to obtain a hierarchical frustum nanopore SERS substrate.

[0021] The preferred technical solution provided by this invention is:

[0022] In step S1, the method for treating the hydrophilicity of the PI membrane is specifically as follows:

[0023] The polyimide (PI) film was cut into 2 cm × 2 cm samples, and then ultrasonically cleaned in anhydrous ethanol for 15 minutes. The PI film was then rinsed with deionized water, air-dried, and finally subjected to a 3-minute surface hydrophilic treatment using a plasma cleaner to obtain a hydrophilic surface.

[0024] Another preferred technical solution provided by the present invention is:

[0025] In step S1, the self-assembly method of monolayer PS microspheres is specifically as follows:

[0026] Take 300 μL of a 5% (w / w) PS microsphere stock solution with a particle size of 6 μm and mix it with anhydrous ethanol at a volume ratio of 3:1 to prepare a PS microsphere ethanol mixture. Add this mixture vertically to a glass petri dish containing deionized water. The PS microspheres self-assemble at the gas-liquid interface to form a monolayer. Add 1 μL (w / w) of surfactant TX-100 to the petri dish. The PS microspheres form a dense monolayer on the surface of the deionized water. Slowly insert the hydrophilically treated PI membrane into the deionized water at a 45° angle and gradually move it below the monolayer of PS microspheres. Then, pull the PI membrane upwards at a constant rate to transfer the monolayer of PS microspheres to the PI surface. After air drying, the monolayer of PS microspheres stably self-assembles on the PI membrane surface.

[0027] The present invention provides a further preferred technical solution as follows:

[0028] The etching parameters for step S2 are as follows: RF power is 50W, inductively coupled plasma power is 150W, vacuum in the process chamber is maintained at 18 to 20 mTorr, oxygen flow rate is 30 sccm, helium flow rate is 50 sccm, and etching time is 30 minutes.

[0029] Preferred,

[0030] The specific steps of step S3, the PDMS molding method, are as follows:

[0031] The PDMS precursor and curing agent were uniformly mixed at a weight ratio of 10:1, and the mixture was evacuated for 15 minutes to remove air bubbles. The mixture was then slowly added to the surface of the etched PI film and cured on a 70°C hot plate for 2 hours. After curing, the PDMS was peeled off to obtain an ordered micron-scale pit array structure.

[0032] In addition,

[0033] Step S4 specifically involves:

[0034] PDMS with an array of pits was treated in a plasma cleaner for 1 minute to obtain a hydrophilic surface. 100 μL of a 5% (w / w) PS nanosphere stock solution with a particle size of 600 nm was mixed with anhydrous ethanol at a 1:1 volume ratio to prepare a PS nanosphere-ethanol mixture. This mixture was then added dropwise to deionized water, where the PS nanospheres formed a dense monolayer on the surface of the deionized water. The hydrophilically treated PDMS was slowly inserted into the deionized water at a 45° angle and gradually moved beneath the monolayer of PS nanospheres. The PDMS was then pulled upwards at a constant rate to transfer the monolayer of PS nanospheres into the pits within the PDMS. After air drying, the monolayer of PS nanospheres stably self-assembled within the PDMS pits.

[0035] Furthermore,

[0036] It also includes step S7, the uniformity test of the substrate Raman spectrum;

[0037] After hydrophilic treatment of the substrate, MGITC was modified on the substrate surface, and the Raman signal of MGITC at different locations was detected for substrate signal uniformity testing.

[0038] Step S7 specifically involves: placing the substrate in a plasma cleaner for 3 minutes, followed by immersion in a plasma cleaning solution with a concentration of 10... -7 The substrate was incubated in MGITC solution for 2 hours under constant temperature and shaking conditions. After being removed, it was rinsed three times with deionized water and allowed to air dry at room temperature. Raman spectroscopy was used to sample different locations on the substrate surface to verify the uniformity of the substrate.

[0039] This invention also provides a technical solution for the application of a SERS substrate prepared by a method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate in a microfluidic chip.

[0040] Furthermore, the specific steps include:

[0041] Step S8, substrate functionalization;

[0042] SERS were hydrophilically treated and then immersed in solutions of lead and hexavalent chromium ion specific recognition molecules, which were then shaken to allow the recognition molecules to fully bond to the substrate surface, thus achieving functional modification of the SERS substrate.

[0043] Step S9: Fabrication of the pump-free microfluidic chip and sealing it with the SERS substrate;

[0044] Design a pump-free microfluidic chip, including a detection chamber that can be simultaneously embedded in two SERS substrates, for the synchronous detection of lead-chromium ions.

[0045] in,

[0046] The specific method for recognizing molecular modifications in step S8 is as follows:

[0047] 250 μL of a 1 μM lead ion-specific aptamer solution and 0.2 M NaCl solution were mixed together in a centrifuge tube. The SERS substrate, which had undergone plasma cleaning and hydrophilization treatment, was then immersed in this mixture and incubated with shaking for 12 hours. The thiol groups at one end of the aptamer were immobilized with gold nanoparticles on the SERS substrate surface via Au-S bonds. The SERS substrate immobilized with the aptamer was rinsed three times with deionized water and then transferred to 500 μL of a 1 μM substrate chain solution. After incubation with shaking for 12 hours, the substrate chain and the immobilized lead ion-specific aptamer underwent complementary hybridization to form a complete lead ion-specific double-stranded aptamer. The substrate was rinsed three times with deionized water, air-dried at room temperature, and then embedded into a microfluidic chip.

[0048] 250 μL of a 1 μM methimazole solution was placed in a centrifuge tube. Another SERS substrate, which had undergone plasma hydrophilic treatment, was immersed in the solution and incubated with shaking for 12 hours. Methimazole was immobilized by forming Au-S bonds with gold nanoparticles on the substrate surface through its thiol groups. The substrate was rinsed three times with deionized water, air-dried at room temperature, and then embedded into a microfluidic chip.

[0049] Preferred,

[0050] The fabrication of the pump-free microfluidic chip and its sealing with the SERS substrate in step S9 includes the following steps:

[0051] Fabrication of the S901 pump-free microfluidic chip: A mask containing two SERS substrates and microfluidic channels was designed using AutoCAD software. A 100 μm thick layer of SU-8 3035 photoresist was spin-coated onto a silicon wafer using a spin coater. The mask was then exposed to 365 nm ultraviolet light using a UV lithography machine to transfer the channel pattern onto the silicon wafer. The patterned silicon wafer was placed in a petri dish. A PDMS precursor and curing agent were mixed at a weight ratio of 10:1, thoroughly stirred, and degassed under vacuum. The mixture was then poured into the petri dish, cured by heating, and then peeled off to form the microfluidic chip. The chip is approximately 44 mm long, 20 mm wide, and 5 mm thick. The channel width is 200 μm and the height is 100 μm. The reserved substrate groove area is 4.3 mm long, 2 mm wide, and 0.2 mm high.

[0052] S902, Hydrophilic treatment of pump-free microfluidic chip: Treat the PDMS microfluidic chip with oxygen plasma for 90 seconds, then place it in a polyethylene glycol (PEG) solution preheated to 150°C for 25 minutes. After cooling to room temperature, rinse the microfluidic chip three times each with isopropanol and deionized water to completely remove residual PEG.

[0053] S903, pump-free microfluidic chip and SERS substrate sealing: The PDMS chip and glass slide are placed together in a plasma cleaner and treated with oxygen plasma for 2 minutes. After that, the PDMS is removed, and the SERS substrate incubated with lead-chromium specific recognition molecules is embedded into the corresponding reserved groove of the PDMS chip. Finally, the glass slide is bonded to the PDMS to complete the encapsulation of the entire microfluidic chip.

[0054] This invention also provides a technical solution for the application of a microfluidic chip fabricated on a hierarchical frustum porous synergistic light-trapping SERS substrate in the trace detection of heavy metal ions, the specific steps of which are as follows:

[0055] Step S10: Add a solution containing lead and hexavalent chromium ions to the prepared chip, incubate statically for 30 minutes, and perform Raman detection using a Raman spectrometer;

[0056] 20 μL of test solutions containing different concentrations of lead ions and hexavalent chromium ions were introduced into different pump-free microfluidic chips. The solutions filled the channels within 40 seconds without external actuation due to capillary action within the microchannels and the pressure generated by the inlet liquid level difference. Afterward, the chips were incubated for 30 minutes to ensure sufficient reaction between the target ions and the specific recognition molecules on the SERS substrate, followed by Raman signal detection using a Raman spectrometer.

[0057] The Raman spectrometer parameters were set as follows: excitation source of 633 nm, laser power of 0.25–0.3 mW, integration time of 2 seconds, and integration count of 3.

[0058] The beneficial effects of this invention are:

[0059] 1. The hierarchical frustum-shaped porous synergistic light-trapping SERS substrate has micron-scale frustum and nano-scale pore structure, which improves the incident light trapping efficiency and plays a key role in improving the local electromagnetic field intensity and enhancing the Raman scattering signal, providing a structural basis for sensitive and efficient SERS detection.

[0060] 2. By integrating the substrate into a pump-free microfluidic chip and combining it with the chip's self-driving capability, it has the advantages of simple operation, small sample volume, and fast response speed, enabling rapid and accurate detection of various heavy metal ions. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the fabrication process of the hierarchical frustum porous synergistic light-trapping SERS substrate described in this invention;

[0062] Figure 2 Photos of the SERS substrate before and after gold plating;

[0063] Figure 3The graph shows the signal uniformity test results of the substrate when detecting MGITC molecules.

[0064] Figure 4 A schematic diagram of the detection mechanism for lead and hexavalent chromium ions;

[0065] Figure 5 The images show the physical diagram of the pump-free microfluidic chip designed for this invention and a schematic diagram of dual metal ion detection.

[0066] Figure 6 The SERS spectra and linear fitting curves of lead and hexavalent chromium ions at different concentrations are shown. Detailed Implementation

[0067] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0068] Figure 1 This is a schematic diagram of the fabrication process of the hierarchical frustum porous synergistic light-trapping SERS substrate described in this invention.

[0069] The substrate is prepared by the following steps:

[0070] Step S1: Monolayer PS microspheres self-assemble on PI film;

[0071] The PI membrane was subjected to hydrophilic treatment using a plasma cleaner. A stock solution of 5% PS microspheres with a particle size of 6 μm was mixed with anhydrous ethanol at a volume ratio of 3:1. The prepared mixture was then added dropwise vertically to a glass petri dish containing deionized water, where the microspheres formed a monolayer membrane structure on the deionized water surface. The monolayer PS microspheres were then transferred from the water surface to the PI membrane surface using a dip-coating method. After natural drying, a PI membrane with a monolayer PS microspheres was obtained.

[0072] Step S2: Form a frustum structure on the PI surface based on ICP etching;

[0073] A PI film with a single layer of PS microspheres was placed in the reaction chamber of an inductively coupled plasma etching machine for oxygen plasma etching. Using the PS microspheres as a mask, a periodic frustum array was formed on the surface of the PI film. The specific etching parameters were: RF power of 50W, inductively coupled plasma power of 150W, vacuum in the process chamber maintained at 18 to 20 mTorr, oxygen flow rate of 30 sccm, helium flow rate of 50 sccm, and etching time of 30 minutes.

[0074] Step S3: Toluene is used to dissolve and remove the residual PS microsphere template, and a micron-scale pit array is formed by molding with PDMS.

[0075] The etched PI film was placed in toluene solution for 10 minutes to dissolve the residual PS microspheres. PDMS was poured onto the surface of the PI film and placed on a 70°C heating plate for 2 hours. After the PDMS was completely cured, it was peeled off from the PI film to obtain a PDMS pit array.

[0076] Step S4: Monolayer PS nanospheres are self-assembled in micron-sized pits and SU-8 photoresist is dropped onto the nanosphere array to cover it;

[0077] After hydrophilic treatment, PDMS with pits is transferred into the pits by a Czochralski method, and SU-8 photoresist is poured onto the PS nanospheres and the surface of the PDMS pits.

[0078] Step S5: Crosslink SU-8 with ultraviolet light and remove nanospheres to form nanopores;

[0079] After being exposed and cured by 365 nm ultraviolet light, the SU-8 photoresist was peeled off from the PDMS, and the cured photoresist was immersed in toluene solution for 10 minutes to remove the PS nanospheres and form a nanoporous structure on the surface of the frustum.

[0080] Step S6: Electron beam evaporation deposits a gold film. The actual substrate image is shown below. Figure 2 As shown;

[0081] The prepared frustum nanopore array photoresist substrate was placed in the chamber of a vacuum deposition system, and a 30 nm gold film was uniformly deposited under high vacuum conditions at a deposition rate of 0.02 nm / s to 0.03 nm / s to obtain a hierarchical frustum nanopore SERS substrate.

[0082] Step S7, uniformity test of the substrate Raman spectrum, the detection results are as follows: Figure 3 As shown;

[0083] The substrate was placed in a plasma cleaner for 3 minutes, and then immersed in a 10% concentration plasma cleaner. -7 The substrate was incubated in MGITC solution under constant temperature and shaking conditions for 2 hours. After removal, it was rinsed three times thoroughly with deionized water and allowed to air dry at room temperature. Raman spectroscopy was used to sample different locations on the substrate surface to verify the homogeneity of the substrate. The spectrum was calculated at 1614 cm⁻¹. -1 The relative standard deviation is 4.08%, indicating good homogeneity of the substrate;

[0084] The specific steps for using the hierarchical frustum porous synergistic light-trapping SERS substrate of the present invention to fabricate microfluidic chips are as follows:

[0085] Step S8, substrate functionalization;

[0086] 250 μL of a 1 μM lead ion-specific aptamer solution and 0.2 M NaCl solution were mixed together in a centrifuge tube. The SERS substrate, which had undergone plasma cleaning and hydrophilization treatment, was then immersed in this mixture and incubated with shaking for 12 hours. The thiol groups at one end of the aptamer were immobilized with gold nanoparticles on the SERS substrate surface via Au-S bonds. The SERS substrate immobilized with the aptamer was rinsed three times with deionized water and then transferred to 500 μL of a 1 μM substrate chain solution. After incubation with shaking for 12 hours, the substrate chain and the immobilized lead ion-specific aptamer underwent complementary hybridization to form a complete lead ion-specific double-stranded aptamer. The substrate was rinsed three times with deionized water, air-dried at room temperature, and then embedded into a microfluidic chip.

[0087] 250 μL of a 1 μM methimazole solution was placed in a centrifuge tube. Another SERS substrate, which had undergone plasma hydrophilic treatment, was immersed in the solution and incubated with shaking for 12 hours. Methimazole was immobilized by forming Au-S bonds with gold nanoparticles on the substrate surface through its thiol groups. The substrate was rinsed three times with deionized water, air-dried at room temperature, and then embedded into a microfluidic chip.

[0088] Step S9: Fabrication of the pump-free microfluidic chip and sealing it with the SERS substrate. A photograph of the completed sealing process is shown below. Figure 5 As shown;

[0089] The process is divided into the following three steps:

[0090] Fabrication of the S901 pump-free microfluidic chip: A mask containing two SERS substrates and microfluidic channels was designed using AutoCAD software. A 100 μm thick layer of SU-8 3035 photoresist was spin-coated onto a silicon wafer using a spin coater. The mask was then exposed to 365 nm ultraviolet light using a UV lithography machine to transfer the channel pattern onto the silicon wafer. The patterned silicon wafer was placed in a petri dish. A PDMS precursor and curing agent were mixed at a weight ratio of 10:1, thoroughly stirred, and degassed under vacuum. The mixture was then poured into the petri dish, cured by heating, and then peeled off to form the microfluidic chip. The chip is approximately 44 mm long, 20 mm wide, and 5 mm thick. The channel width is 200 μm and the height is 100 μm. The reserved substrate groove area is 4.3 mm long, 2 mm wide, and 0.2 mm high.

[0091] S902, Hydrophilic treatment of pump-free microfluidic chip: Treat the PDMS microfluidic chip with oxygen plasma for 90 seconds, then place it in a polyethylene glycol (PEG) solution preheated to 150°C for 25 minutes. After cooling to room temperature, rinse the microfluidic chip three times each with isopropanol and deionized water to completely remove residual PEG.

[0092] S903, pump-free microfluidic chip and SERS substrate sealing: The PDMS chip and glass slide are placed together in a plasma cleaner and treated with oxygen plasma for 2 minutes. After that, the PDMS is removed, and the SERS substrate incubated with lead-chromium specific recognition molecules is embedded into the corresponding reserved groove of the PDMS chip. Finally, the glass slide is bonded to the PDMS to complete the encapsulation of the entire microfluidic chip.

[0093] The specific method for using the microfluidic chip prepared above for trace detection of heavy metal ions is as follows:

[0094] Step S10, Trace detection of heavy metal ions, the SERS detection principle of lead and hexavalent chromium ions is as follows: Figure 4 As shown;

[0095] 20 μL of test solutions containing different concentrations of lead and hexavalent chromium ions were introduced into different pump-free microfluidic chips. The solutions filled the channels within 40 seconds without external actuation due to capillary action and pressure generated by the inlet liquid level difference. Afterward, the chips were incubated for 30 minutes to ensure sufficient reaction between the target ions and the specific recognition molecules on the SERS substrate. Raman signals were then detected using Raman spectroscopy. The SERS spectra and linear fitting curves for lead and hexavalent chromium ions at different concentrations are shown below. Figure 6 As shown in the figure, the detection limit for lead and hexavalent chromium ions of this invention can reach 10. -11 and 10 -10 M.

[0096] The Raman spectrometer parameters were set as follows: excitation source of 633 nm, laser power of 0.25–0.3 mW, integration time of 2 seconds, and integration count of 3.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate, characterized in that, Includes the following steps: Step S1: Monolayer PS microspheres self-assemble on PI film; The PS microsphere stock solution was mixed with anhydrous ethanol, and the mixture was dropped into a culture dish containing deionized water. The microspheres self-assembled on the water surface to form a monolayer under the action of surface tension. The PI membrane is hydrophilicized, and the monolayer PS microspheres on the water surface are transferred onto the PI membrane by the dip-coating method. After natural air drying, a PI film with a single layer of PS microspheres is obtained; Step S2: Form a frustum structure on the PI surface based on ICP etching; The PI film with monolayer PS microspheres obtained in step S1 is placed in the chamber of an inductively coupled plasma etching machine for etching, forming a periodic frustum array on the surface of the PI film. Step S3: Toluene is used to dissolve and remove the residual PS microsphere template, and a micron-scale pit array is formed by molding with PDMS. The substrate prepared in step S2 was immersed in toluene solution to dissolve the incompletely etched PS microspheres. Polydimethylsiloxane (PDMS) was used for molding, and an ordered pit array was obtained after the PDMS was completely cured. Step S4: Monolayer PS nanospheres are self-assembled in micron-sized pits and SU-8 photoresist is dropped onto the nanosphere array to cover it; After the pit array prepared in step S3 is hydrophilically treated, the monolayer PS nanospheres are transferred from the surface of deionized water to the inside of the pits by the dip-coating method, and photoresist is poured onto the surface of PDMS and PS nanospheres. Step S5: Crosslink SU-8 with ultraviolet light and remove nanospheres to form nanopores; After UV curing, the cured SU-8 photoresist was peeled off and soaked in toluene solution to dissolve the PS nanospheres, forming a porous frustum structure on the photoresist surface. Step S6: Electron beam evaporation deposits a gold film; The porous frustum photoresist substrate obtained in step S5 is placed in the chamber of a vacuum coating instrument to deposit a gold film. Finally, gold nanoparticles are formed on the sidewalls of the frustum and inside the nanopores to obtain a hierarchical frustum porous synergistic light-trapping SERS substrate, thus completing the preparation of the hierarchical frustum porous synergistic light-trapping SERS substrate.

2. The method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate according to claim 1, characterized in that, In step S1, the self-assembly method of monolayer PS microspheres is specifically as follows: Take 300 μL of PS microsphere stock solution with a mass fraction of 5% and a particle size of 6 μm, mix it with anhydrous ethanol at a volume ratio of 3:1 to prepare PS microsphere ethanol mixture, and add the mixture dropwise into a glass culture dish containing deionized water in a vertical direction. PS microspheres self-assemble at the gas-liquid interface to form a monolayer film. Add 1 μL of 2% (v / v) surfactant TX-100 to a petri dish, and PS microspheres will form a dense monolayer on the surface of deionized water; The hydrophilic PI membrane was slowly inserted into deionized water at a 45° angle and gradually moved to the bottom of the monolayer PS microspheres. Then, the PI membrane was pulled upward at a constant rate to transfer the monolayer PS microspheres to the PI surface. After natural air drying, the monolayer PS microspheres stably self-assemble on the PI film surface.

3. The method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate according to claim 1, characterized in that, The etching parameters for step S2 are as follows: The radio frequency (RF) power was set to 50W, the inductively coupled plasma (ICP) power was set to 150W, the vacuum in the process chamber was maintained at 18 to 20 mTorr, the oxygen flow rate was 30 sccm, the helium flow rate was 50 sccm, and the etching process lasted for 30 minutes.

4. The method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate according to claim 1, characterized in that, In step S4, the self-assembly method of monolayer PS nanospheres is specifically as follows: PDMS with a pitted array was placed in a plasma cleaner for 1 minute to obtain a hydrophilic surface; Take 100 μL of PS nanosphere stock solution with a mass fraction of 5% and a particle size of 600 nm, mix it with anhydrous ethanol at a volume ratio of 1:1 to prepare PS nanosphere ethanol mixture, add the mixture dropwise to deionized water, and PS nanospheres form a dense monolayer on the surface of deionized water. The hydrophilically treated PDMS was slowly inserted into deionized water at a 45° angle and gradually moved to the bottom of the monolayer PS nanospheres. Then, the PDMS was pulled upward at a constant rate to transfer the monolayer PS nanospheres into the PDMS pits. After air drying, the monolayer PS nanospheres stably self-assemble inside the PDMS pits.

5. The method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate according to claim 1, characterized in that, It also includes step S7, the uniformity test of the substrate Raman spectrum; After hydrophilic treatment of the substrate, MGITC was modified on the substrate surface, and the Raman signal of MGITC at different locations was detected for substrate signal uniformity testing. Specifically, the substrate is placed in a plasma cleaner for 3 minutes, and then immersed in a solution with a concentration of 10... -7 The substrate was incubated in MGITC solution for 2 hours under constant temperature and shaking conditions. After being removed, it was rinsed three times with deionized water and allowed to air dry at room temperature. Raman spectroscopy was used to sample different locations on the substrate surface to verify the uniformity of the substrate.

6. The application of the SERS substrate prepared by the method for preparing a hierarchical frustum porous synergistic light-trapping SERS substrate according to claims 1-5 in microfluidic chips.

7. The application of the hierarchical frustum porous synergistic light-trapping SERS substrate according to claim 6 in microfluidic chips, characterized in that, The specific steps are as follows: Step S8, substrate functionalization; SERS were hydrophilically treated and then immersed in solutions of lead and hexavalent chromium ion specific recognition molecules, which were then shaken to allow the recognition molecules to fully bond to the substrate surface, thus achieving functional modification of the SERS substrate. Step S9: Fabrication of the pump-free microfluidic chip and sealing it with the SERS substrate; Design a pump-free microfluidic chip, including a detection chamber that can be simultaneously embedded in two SERS substrates, for the synchronous detection of lead-chromium ions.

8. The application of the hierarchical frustum porous synergistic light-trapping SERS substrate according to claim 7 in microfluidic chips, characterized in that, The specific method for recognizing molecular modifications in step S8 is as follows: Take 250 μL of a 1 μM lead ion-specific aptamer solution and 0.2 M NaCl solution, add them together into a centrifuge tube and mix. Immerse the SERS substrate, which has been hydrophilized by a plasma cleaner, in the above mixture and shake and incubate for 12 hours. The thiol group at one end of the aptamer is fixed to the gold nanoparticles on the surface of the SERS substrate through Au-S bonds. After rinsing the SERS substrate with the aptamer three times with deionized water, it was transferred to 500 μL of substrate chain solution with a concentration of 1 μM and incubated with shaking for 12 hours to allow the substrate chain to hybridize with the immobilized lead ion-specific aptamer to form a complete lead ion-specific double-stranded aptamer. Rinse the substrate three times with deionized water and air dry at room temperature; Methimazole was used as a chromium ion specific recognition solution. 250 μL of 1 μM methimazole solution was placed in a centrifuge tube, and another SERS substrate that had been hydrophilically treated by plasma was immersed in the solution and incubated with shaking for 12 hours. Methimazole was fixed by forming Au-S bonds with gold nanoparticles on the substrate surface through thiol groups. Rinse the substrate three times with deionized water and air dry at room temperature.

9. The application of the hierarchical frustum porous synergistic light-trapping SERS substrate according to claim 8 in a microfluidic chip, characterized in that, Step S9 specifically includes the following steps: S901, Fabrication of a pump-free microfluidic chip: A mask containing two SERS substrates and microfluidic channels was designed using AutoCAD software; A 100 μm thick layer of SU-8 3035 photoresist was spin-coated onto a silicon wafer using a spin coater. The photomask was then exposed to 365 nm ultraviolet light using an ultraviolet lithography machine to transfer the channel pattern onto the silicon wafer. Place the patterned silicon wafer into a petri dish; The PDMS precursor and curing agent were mixed at a weight ratio of 10:1, stirred thoroughly, and degassed under vacuum. The mixture was then poured into a petri dish, heated and cured, and then peeled off to form a microfluidic chip. S902, hydrophilic treatment of pump-free microfluidic chip: The PDMS microfluidic chip was treated with oxygen plasma for 90 seconds, and then placed in a polyethylene glycol (PEG) solution preheated to 150°C for 25 minutes. After cooling to room temperature, the microfluidic chip was rinsed three times each with isopropanol and deionized water to completely remove residual PEG. S903, pump-free microfluidic chip and SERS substrate bonding: The PDMS chip and glass slide were placed together in a plasma cleaner and treated with oxygen plasma for 2 minutes. Then, the PDMS was removed, and the SERS substrate incubated with lead-chromium specific recognition molecules was embedded into the corresponding reserved groove of the PDMS chip. Finally, the glass slide was attached to the PDMS to complete the packaging of the entire microfluidic chip.

10. An application of a hierarchical frustum porous synergistic light-trapping SERS substrate as described in any one of claims 6-9 in a microfluidic chip, characterized in that, Including step S10, trace detection of heavy metal ions; 20 μL of test solutions containing different concentrations of lead ions and chromium ions were introduced into different pump-free microfluidic chips. The solution can fill the flow channel within 40 seconds without external drive, thanks to the capillary action within the microchannel and the pressure generated by the inlet liquid level difference. After incubation for 30 minutes to ensure that the target ions fully react with the specific recognition molecules on the SERS substrate, the Raman signal is then detected by Raman spectroscopy.

Images