Preparation method of novel composite honeycomb-shaped SERS (Surface Enhanced Raman Scattering) substrate and application of novel composite honeycomb-shaped SERS substrate combined with micro-fluidic chip in trace detection

Abstract

The invention discloses a preparation method of a novel composite honeycomb-shaped SERS (Surface Enhanced Raman Scattering) substrate and application of the SERS substrate in trace detection in combination with a micro-fluidic chip. The substrate has the advantages of high sensitivity, good repeatability, strong stability and the like. The method comprises the following steps: firstly, preparing a compactly arranged multi-layer polystyrene (PS) microsphere template, and removing the PS microsphere template by a sacrificial template method to form a honeycomb porous skeleton structure taking zinc oxide as a base material; according to the method, a honeycomb-shaped porous framework structure is used as a framework, and zinc oxide nanowires grow on the honeycomb-shaped porous framework structure. The SERS performance of the substrate is enhanced by plating gold on the surface of the substrate through electron beam evaporation. According to the novel composite honeycomb-shaped SERS substrate provided by the invention, a method of combining'structure compounding 'and'material compounding' is adopted, so that the SERS performance of the substrate is remarkably improved, a great specific surface area is provided, and more detection sites are provided for molecules to be detected. The composition of the structure is reflected in the composition of a honeycomb-shaped porous skeleton structure and a nanowire structure; the compounding of the material is the compounding between zinc oxide and gold. The composition of the structure and the material synergistically enhances SERS signals from two aspects of physical enhancement and chemical enhancement. The method has the advantages of being high in sensitivity, good in repeatability, high in stability and the like, and a feasible method is provided for rapid and sensitive detection of trace substances.

Description

Technical Field

[0001] The present invention belongs to the field of micro-nano sensing and microfluidics. Specifically, the present invention relates to a preparation method of a novel composite honeycomb SERS substrate and its application in trace detection in combination with a microfluidics chip. Background Art

[0002] As a vibrational spectroscopy technique, Raman spectroscopy can provide unique fingerprint information of molecules. However, its application faces a fundamental challenge: the non-resonant Raman scattering efficiency generated by the direct interaction between light and molecules is extremely low, and the number of scattered photons is only about one millionth of the incident photons (about 10 -6 ), resulting in a very small Raman scattering cross section. Therefore, the Raman signal detected in the experiment is usually extremely weak or even difficult to capture. Although resonant Raman scattering occurs when the energy of the molecular excited state is low, resulting in a resonant Raman spectrum, this process is often limited by interference factors such as fluorescence emission, which greatly restricts the application range of Raman spectroscopy. The surface-enhanced Raman scattering (SERS) effect can significantly amplify the Raman signal of molecules adsorbed on the surface of noble metals, thereby greatly improving the detection sensitivity of Raman spectroscopy. At present, spectral methods based on SERS have achieved single-molecule level detection, and single-molecule surface-enhanced Raman spectroscopy technology has been developed. Early SERS research mainly relied on metal substrates, using surface resonance induced by excitation light to enhance the Raman signal. In order to pursue a stronger enhancement effect and simultaneously improve the optical, electrical properties, stability and erasability of the substrate, the research field has gradually expanded to semiconductor materials. Simulation calculation analysis shows that the surface of semiconductor materials does not have localized surface plasmon resonance characteristics similar to those of noble metals, which means that its Raman enhancement effect does not originate from plasmons. Numerous studies have attributed the enhancement mechanism of semiconductor substrates to a chemical mechanism, namely, the charge transfer mechanism. When a semiconductor material is used as an enhancement substrate, it facilitates charge transfer between the substrate surface and the molecules being detected. Currently, an increasing number of semiconductor nanomaterials (such as the common Fe2O3, CdS, ZnO, CuO, Cu2O, SiO2, TiO2, etc.) are being widely researched and developed as SERS substrates. Summary of the Invention

[0003] The purpose of the present invention is to provide a novel composite honeycomb SERS substrate. Based on the composite honeycomb nanostructure and the electron transfer effect between the semiconductor material zinc oxide and the precious metal gold, a highly sensitive novel SERS substrate is prepared. The present invention first uses a sacrificial template method using PS microspheres to prepare a porous zinc oxide skeleton structure. Zinc oxide nanowires are then grown on the porous zinc oxide skeleton using a hydrothermal method. A 30-60 nm gold film is then deposited using a vacuum coating apparatus, ultimately yielding a novel composite honeycomb SERS substrate. The SERS substrate prepared by this invention exhibits the advantages of high sensitivity, good repeatability, and stable properties, providing a feasible method for the rapid and sensitive detection of trace substances.

[0004] To achieve the above object, the technical solution provided by the present invention is:

[0005] A method for preparing a novel composite honeycomb SERS substrate is characterized by comprising the following steps:

[0006] Step S1, preparing a novel composite honeycomb SERS substrate;

[0007] First, a uniformly packed array of PS microspheres is deposited on a glass slide as a template. A zinc oxide precursor solution is then immersed into the prepared PS microsphere template. The template is then annealed at high temperature to remove the PS spheres, resulting in a honeycomb-like porous framework. The prepared honeycomb porous framework is then immersed in a prepared zinc oxide precursor solution. A zinc oxide nanowire secondary structure is then hydrothermally formed on the honeycomb porous framework prepared in the previous step. Finally, a gold film is evaporated onto the substrate using an electron beam vacuum coating system to complete the substrate preparation.

[0008] The step S1 is specifically as follows:

[0009] Step S1.1, preparing a mixed solution of zinc oxide precursor solution and PS microspheres;

[0010] An aqueous solution or ethanol solution of zinc nitrate hexahydrate and citric acid with a mass fraction of 25% to 35% was prepared. An appropriate amount of zinc nitrate hexahydrate powder was first weighed and dissolved in water at room temperature. Then, citric acid powder in an equal molar ratio to zinc ions was slowly added to the prepared zinc nitrate aqueous solution or ethanol solution. The mixture was stirred continuously until the solution clarified to obtain a zinc oxide precursor solution. PS microspheres with a diameter of 2 μm were then doped into the prepared zinc oxide precursor solution to prepare a mixed solution of the zinc oxide precursor solution and PS microspheres.

[0011] Step S1.2, preparation of multilayer PS microsphere template;

[0012] First, clean the glass slides. The cut glass slides are ultrasonically cleaned in anhydrous ethanol and then deionized water to remove the organic coating on the surface. The cleaned glass slides are then treated with oxygen plasma in a plasma cleaner for 2 minutes to increase their hydrophilicity. The slides are then vertically immersed in a mixture of the zinc oxide precursor solution and PS microspheres prepared in the previous step. The slides are then transferred to a 70°C oven. As the solvent evaporates, the PS microspheres form a uniform, dense multilayer structure on the glass slide due to surface tension.

[0013] Step S1.3, preparation of a honeycomb porous framework;

[0014] The glass sheet with the multi-layer PS sphere template deposited was taken out from the oven, transferred to a muffle furnace and heated at a constant rate to 450-500°C and maintained for 1.5-2.5 hours to remove the PS microsphere template and form a stable zinc oxide honeycomb structure between the original PS sphere templates.

[0015] Step S1.4, preparation of secondary structure of zinc oxide nanowires;

[0016] First, a zinc oxide precursor solution of zinc nitrate and hexamethylenetetramine is prepared. The honeycomb-shaped porous skeleton prepared in step S1.3 is treated with oxygen plasma to increase its hydrophilicity, and then vertically immersed in the solution and transferred to a reactor. The skeleton is placed in an oven for heating. After the reactor cools to room temperature, the substrate is taken out and rinsed in running water to remove the large-sized zinc oxide particles attached to the surface. Finally, the skeleton is placed on a clean laboratory bench and naturally air-dried to obtain a composite honeycomb-shaped three-dimensional hierarchical structure with uniform size and good stability.

[0017] Step S1.5, gold plating on the substrate surface;

[0018] The substrate is gold-coated using a vacuum coater. The sample stage with the substrate sample attached is placed upside down in the chamber of the vacuum coater. The vacuum pump is activated to reduce the chamber pressure to below 5 × 10⁻⁴ Pa. The electron beam source is then powered on, the filament preheated for 2 minutes, and the beam spot adjusted to align with the center of the target. Once the beam current stabilizes at the set value, the sample shutter is opened and a gold film is evaporated onto the substrate at a constant rate. When the film thickness reaches 30–60 nm, evaporation is stopped and the electron beam source is turned off. Finally, the chamber is depressurized to produce a novel composite honeycomb SERS substrate.

[0019] Step S2, preparation of microfluidic chip;

[0020] The microfluidic chip mask pattern required for this invention was first designed in AutoCAD modeling software. A professional processing method was used to obtain the mask pattern for photolithography. Then, photolithography was used to prepare the positive template for the microfluidic chip. This template was then molded with polydimethylsiloxane (PDMS). After the PDMS solidified, holes were drilled and cleaned. Finally, the microfluidic chip used in this experiment was sealed with a glass slide.

[0021] The step S2 is specifically as follows:

[0022] This study fabricated a microfluidic chip based on a mask pattern designed in AutoCAD. First, a positive template with a 30μm channel height was prepared. The silicon wafer was cleaned with alcohol and pre-baked at 105°C for 10 minutes. SU8-3025 photoresist was spin-coated. A pre-baking process was performed at 95°C for 12 minutes, followed by three exposures. A post-baking process was performed at 95°C for 3 minutes to visualize the pattern. The positive template was then developed with ethyl lactate for 5 minutes. The presence of white flocs was confirmed by isopropyl alcohol testing, and the template was dried with nitrogen.

[0023] The PDMS chip was then prepared by mixing a prepolymer and curing agent in a ratio of 10:1. After degassing, the mixture was cast onto a positive template fixed to a culture dish, while also embedding a stainless steel gasket. After heating to remove bubbles and calibrate the gasket position, the chip was cured at 70°C for 2 hours. After peeling the PDMS, the channel area was excised, and the gasket was removed to form a base groove. After trimming, the catheter interface was prepared using a hole punch.

[0024] Finally, the chip is sealed: the PDMS is ultrasonically cleaned with anhydrous ethanol for 15 minutes, thoroughly dried, and surface impurities are removed. The aptamer-modified substrate is inserted into the groove and treated with oxygen plasma to activate the surface along with the glass slide. The PDMS and glass are then sealed together, ultimately creating a microfluidic chip with an integrated detection substrate.

[0025] Step S3, SERS detection of trace molecules on the microfluidic chip;

[0026] The substrate is embedded in a microfluidic chip, the test liquid is passed into the microfluidic chip, and Raman detection is performed using a Raman spectrometer.

[0027] The step S3 is specifically as follows:

[0028] The substrate was embedded in a microfluidic chip, the test liquid was passed into the microfluidic chip, the chip was placed on a Renishaw Raman test sample stage, and the laser was focused on the composite honeycomb SERS substrate. The spectrometer parameters were set as follows: excitation light source of 633 nm, 50× microscope objective, laser power of 5 mW, integration time of 5 s, and integration times of 2.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention designs a novel composite honeycomb SERS substrate. The composite honeycomb structure can be prepared in batches by using a sacrificial template method combined with a hydrothermal method. The novel composite honeycomb SERS substrate adopts a method combining structural composite and material composite to synergistically enhance the SERS signal of the substrate from the aspects of electromagnetic field enhancement and chemical enhancement, and provides a large specific surface area to provide more detection sites for the molecules to be detected. The composite honeycomb three-dimensional structure can effectively increase the contact between the molecules to be detected and the substrate. The substrate of the present invention can be produced in large quantities, has high repeatability, and is structurally stable. The prepared substrate has good Raman scattering enhancement effect, strong stability, and high sensitivity, and can be used for high-sensitivity detection of trace molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart for the preparation of a novel composite honeycomb SERS substrate of the present invention;

[0032] Figure 2 for Figure 1 Scanning electron microscope image of the honeycomb skeleton structure;

[0033] Figure 3 for Figure 1 Scanning electron microscope image of the new composite honeycomb SERS substrate;

[0034] Figure 4 This is an electron microscope image of a microfluidic chip (bright red ink);

[0035] Figure 5 This is a test of the uniformity of the SERS signal of the substrate of the present invention;

[0036] Figure 6 The SERS enhancement comparison between the honeycomb skeleton structure gold-plated substrate and the composite honeycomb SERS substrate;

[0037] Figure 7 Detection limit verification diagram of the SERS substrate of the present invention; DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings.

[0039] Figure 1 This is a flow chart for preparing the novel composite honeycomb SERS substrate of the present invention.

[0040] Step S1, preparing a composite honeycomb SERS substrate;

[0041] First, a uniformly packed array of PS microspheres is deposited on a glass slide as a template. A zinc oxide precursor solution is then immersed into the prepared PS microsphere template. The template is then annealed at high temperature to remove the PS spheres, resulting in a honeycomb-like porous framework. The prepared honeycomb porous framework is then immersed in a prepared zinc oxide precursor solution. A secondary structure of zinc oxide nanowires is hydrothermally grown on the honeycomb porous framework prepared in the previous step. Finally, a gold film is evaporated onto the substrate surface using an electron beam vacuum coating system, completing the substrate preparation.

[0042] The step S1 is specifically as follows:

[0043] Step S1.1, preparing a zinc oxide precursor solution-PS microsphere mixed solution;

[0044] An aqueous solution or ethanol solution of zinc nitrate hexahydrate and citric acid with a mass fraction of 25% to 35% was prepared. An appropriate amount of zinc nitrate hexahydrate powder was first weighed and dissolved in water at room temperature. Then, citric acid powder in an equal molar ratio to zinc ions was slowly added to the prepared zinc nitrate aqueous solution or ethanol solution. The mixture was stirred for 1 hour. After the solution was clarified, a zinc oxide precursor solution was obtained. PS microspheres with a diameter of 2 μm were then doped into the prepared zinc oxide precursor solution to prepare a mixed solution of zinc oxide precursor solution and PS microspheres.

[0045] Step S1.2, preparation of multilayer PS microsphere template;

[0046] First, clean the glass slides. The cut glass slides are ultrasonically cleaned in anhydrous ethanol and then deionized water to remove the organic coating. The cleaned glass slides are then placed in a plasma cleaner and treated with oxygen plasma to increase their hydrophilicity. The slides are then vertically immersed in a mixture of the zinc oxide precursor solution and PS microspheres prepared in the previous step. The slides are then transferred to an oven. As the solvent evaporates, the PS microspheres form a uniform, dense multilayer structure on the glass slide due to surface tension.

[0047] Step S1.3, preparation of a honeycomb porous framework;

[0048] The glass sheet with the multi-layer PS ball template deposited on it was removed from the oven and transferred to a muffle furnace and heated at a constant rate to 450-500°C for 1.5-2.5 hours to remove the PS microsphere template and form a stable zinc oxide honeycomb structure between the original PS ball templates. Figure 2 shown.

[0049] Step S1.4, preparation of secondary structure of zinc oxide nanowires;

[0050] First, a zinc oxide precursor solution of zinc nitrate and hexamethylenetetramine was prepared. The honeycomb-shaped porous skeleton prepared in step S1.3 was treated with oxygen plasma for 2 minutes to increase its hydrophilicity, and then vertically immersed in the solution and transferred to a reactor. The reaction vessel was placed in an oven for heating. After the reactor was cooled to room temperature, the substrate was taken out and rinsed in running water to clean off the large-sized zinc oxide particles attached to the surface. Finally, it was placed on a clean laboratory bench and naturally air-dried to obtain a composite honeycomb-shaped three-dimensional hierarchical structure with uniform size and good stability.

[0051] Step S1.5, gold plating on the substrate surface;

[0052] The substrate is coated with gold on its surface through a vacuum coating instrument, and the sample stage with the substrate sample is inverted in the chamber of the vacuum coating instrument. The vacuum pump is started to pump the chamber pressure to less than 5×10-4Pa. Then the electron beam source power is turned on, the filament is preheated for 2 minutes and the light spot is adjusted to align it with the center of the target. After the beam current stabilizes at the set value, the sample baffle is opened, and a gold film is evaporated on the surface of the substrate at a constant rate. When the film thickness reaches 30 to 60 nm, the evaporation is stopped immediately and the electron beam source is turned off. Finally, the chamber is depressurized to obtain a new composite honeycomb SERS substrate. The electron microscope image of the new composite honeycomb SERS substrate is shown below. Figure 3 shown.

[0053] Step S2, preparation of microfluidic chip;

[0054] The microfluidic chip mask pattern required for this invention was first designed in AutoCAD modeling software. A professional processing method was used to obtain the mask pattern for photolithography. Then, photolithography was used to prepare the positive template for the microfluidic chip. This template was then molded with polydimethylsiloxane (PDMS). After the PDMS solidified, holes were drilled and cleaned. Finally, the microfluidic chip used in this experiment was sealed with a glass slide.

[0055] The step S2 is specifically as follows:

[0056] This study fabricated a microfluidic chip based on a mask pattern designed in AutoCAD. First, a positive template with a 30μm channel height was prepared. The silicon wafer was cleaned with alcohol and pre-baked at 105°C for 10 minutes. SU8-3025 photoresist was spin-coated. A pre-baking process was performed at 95°C for 12 minutes, followed by three exposures. A post-baking process was performed at 95°C to visualize the pattern. Development was performed with ethyl lactate, and complete development was confirmed by detecting white floccules with isopropyl alcohol. The template was then dried with nitrogen to obtain the positive microchannel template.

[0057] The PDMS chip was then prepared by mixing a prepolymer and curing agent in a ratio of 10:1. After degassing, the mixture was cast onto a positive template fixed to a culture dish, while also embedding a stainless steel gasket. After heating to remove bubbles and calibrate the gasket position, the chip was cured at 70°C for 2 hours. After peeling the PDMS, the channel area was excised, and the gasket was removed to form a base groove. After trimming, the catheter interface was prepared using a hole punch.

[0058] Finally, the chip was sealed: PDMS was ultrasonically cleaned with anhydrous ethanol for 15 minutes, and then completely dried to remove surface impurities. The aptamer-modified substrate was embedded in the groove and treated with oxygen plasma for 2 minutes together with the glass slide to activate the surface. The PDMS and glass were then sealed, and the microfluidic chip with integrated detection substrate was finally produced. In order to clearly show the shape of the microfluidic chip channel, the electron microscope image of the microfluidic chip after red ink was introduced into the chip is shown below. Figure 4 shown.

[0059] Step S3, SERS detection of the molecule to be tested on the microfluidic chip;

[0060] The substrate is embedded in a microfluidic chip, the test liquid is passed into the microfluidic chip, and Raman detection is performed using a Raman spectrometer.

[0061] The step S3 is specifically as follows:

[0062] The substrate was embedded in a microfluidic chip, the test liquid was passed into the microfluidic chip, the chip was placed on a Renishaw Raman test sample stage, and the laser was focused on the composite honeycomb SERS substrate. The spectrometer parameters were set as follows: excitation light source of 633 nm, 50× microscope objective, laser power of 5 mW, integration time of 5 s, and integration times of 2.

[0063] Figure 5 To verify the performance of the SERS substrate, Figure 5 A Raman reporter molecule MGITC was used at a concentration of 10 -6 The SERS signal was detected at M, and the SERS signal waterfall diagram proved that the substrate had good uniformity. Figure 6 The composite honeycomb SERS substrate and the honeycomb SERS substrate without zinc oxide nanorod structure were subjected to the same concentration of MGITC (10 -6 M) detection, which proves that the composite honeycomb SERS substrate has better SERS enhancement effect.

[0064] MGITC was selected as the Raman reporter molecule. In aqueous solution, MGITC formed Au-S bonds with the gold on the substrate surface through the thiol group and thus combined with the substrate. -6 ~10 -12 mol / L MGITC solution was mixed, and the substrates bound to different concentrations of MGITC were detected according to the operation of step S3. Figure 7 As shown in the figure, it can be concluded that the SERS enhancement substrate we designed can reach 10 -11 The detection limit of mol / L was 0.05 mol / L, and the substrate had a good enhancement effect and an excellent SERS enhanced detection limit.

[0065] 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 novel composite honeycomb SERS substrate and its application in trace detection in combination with a microfluidic chip, characterized in that: The following steps are included: Step S1, preparing a novel composite honeycomb SERS substrate; Using uniformly packed PS microspheres as templates, a sacrificial template method was used to create a honeycomb-like porous framework with 3 to 6 layers. A secondary structure of zinc oxide nanowires was then grown on the honeycomb porous framework using a hydrothermal method. Finally, a gold film was evaporated onto the substrate using an electron beam vacuum coating system, completing the substrate preparation. Step S2, preparation of microfluidic chip; The microfluidic chip mask pattern required for this invention was designed in AutoCAD modeling software. A high-precision inkjet printer was used to print the photolithographic mask pattern. A positive template for the microfluidic chip was prepared using soft lithography. This template was then molded with polydimethylsiloxane (PDMS). After the PDMS solidified, holes were drilled and cleaned. Finally, the microfluidic chip used in this experiment was sealed with a glass slide. Step S3, SERS detection of trace substances; The novel composite honeycomb SERS substrate prepared in step S1 is sealed with the microfluidic chip prepared in step S2 for SERS detection.

2. The method for preparing a novel composite honeycomb SERS substrate according to claim 1, wherein: The step S1 is specifically as follows: Step S1.1, preparing a mixed solution of zinc oxide precursor solution and PS microspheres; An aqueous solution or ethanol solution of zinc nitrate hexahydrate and citric acid with a mass fraction of 25% to 35% is prepared, and PS microspheres with a diameter of 2 μm are doped into the prepared zinc oxide precursor solution to prepare a mixed solution of the zinc oxide precursor solution and the PS microspheres. Step S1.2, preparation of multilayer PS microsphere template; The cut glass slides were ultrasonically cleaned in anhydrous ethanol and then in deionized water to remove the organic coating. The cleaned glass slides were then treated with oxygen plasma in a plasma cleaner for 1.5–2 minutes to increase their hydrophilicity. The slides were then vertically immersed in a mixture of the zinc oxide precursor solution and PS microspheres prepared in the previous step. The slides were then transferred to an oven. As the solvent evaporated, the PS microspheres formed a uniform, dense multilayer template structure on the glass slide due to surface tension. Step S1.3, preparation of a honeycomb porous framework; The glass sheet with the multi-layer PS sphere template deposited was taken out from the oven, transferred to a muffle furnace and heated at a uniform rate to 450°C to 500°C and maintained for 1.5 to 2.5 hours to remove the PS microsphere template and form a stable zinc oxide honeycomb structure between the original PS sphere templates. Step S1.4, preparation of secondary structure of zinc oxide nanowires; The honeycomb-shaped porous skeleton prepared in step S1.3 was treated with oxygen plasma for 2 minutes to increase its hydrophilicity, and then vertically immersed in a zinc oxide precursor solution and transferred to a reactor. It was placed in a 70°C oven and heated for 3 to 4 hours. After the reactor cooled to room temperature, the substrate was taken out, continuously rinsed in running water, and placed on a clean laboratory bench to dry naturally, thereby obtaining a composite honeycomb-shaped three-dimensional structure with uniform size and good stability. Step S1.5, gold plating on the substrate surface; A 30-60 nm thick gold film was evaporated on the substrate surface using a vacuum coating apparatus to obtain a novel composite honeycomb SERS substrate.

3. The method for preparing a novel composite honeycomb SERS substrate according to claim 1, wherein: The novel composite honeycomb SERS substrate prepared in steps S1.1 to S1.5 utilizes a primary honeycomb porous framework and a secondary nanowire structure to form a honeycomb composite structure. The sacrificial template is prepared by mixing the zinc oxide precursor solution prepared in step S1.1 with PS microspheres, and then vertically depositing the resulting composite template structure of multiple layers of PS microspheres and zinc oxide precursors onto a glass sheet. The honeycomb-like porous skeleton structure prepared in step S1.3 has 3 to 6 layers, and the diameter of the honeycomb-like pores is 1 to 2 μm. The secondary length of the zinc oxide nanowires prepared in step S1.4 is 200 to 500 nm, and the cross-sectional length is 30 to 80 nm. The novel composite honeycomb SERS substrate prepared in step S1.5 has 3 to 6 honeycomb cavity layers, the diameter of the honeycomb holes is 1 to 2 μm, the length of the nanowire structure is 230 to 560 nm, and the thickness of the gold film is 30 to 60 nm.

4. A novel composite honeycomb SERS substrate is combined with a microfluidic chip, and its characteristic is that the novel composite honeycomb SERS substrate is combined with the microfluidic chip for SERS detection.

5. A method for preparing a novel composite honeycomb SERS substrate according to any one of claims 1 to 4 and its application in trace detection.

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