A femtosecond laser-induced plasma SERS substrate preparation method and application

Abstract

The application discloses a preparation method of a femtosecond laser-induced plasma SERS (Surface Enhanced Raman Scattering) substrate, application of the plasma SERS substrate in preparation of a micro-fluidic chip, and trace detection of lead ions in seawater by using the plasma SERS substrate micro-fluidic chip. The application adopts gold-doped nano-particle PMMA (Polymethyl Methacrylate) material, prepares a cylindrical template on a silicon wafer through ultraviolet lithography technology, and pours the gold-doped PMMA into the template to prepare a cylindrical structure. Then, the outer surface of the substrate is roughened by using femtosecond laser modification, and finally, the cylindrical structure is processed into a micro-nano structure with a crater morphology by using femtosecond laser processing technology. The application utilizes the laser-induced plasma expansion principle, breaks through the upper limit of precision of the original micron-level process, realizes structure regulation and control of nanometer-level precision, greatly improves the SERS signal of the substrate, and enhances the detection sensitivity of lead ions.

Description

Technical Field

[0001] The present invention belongs to the field of micro-nano sensing and SERS detection technology, is aimed at detecting lead ions in seawater, and relates to a method and application of preparing a SERS substrate using the principle of femtosecond laser-induced plasma expansion. Background Art

[0002] Raman scattering refers to the phenomenon that the energy of photons changes when they interact with matter. Discovered by Indian physicist C.V. Chandrasekhar Raman (CV Raman) in 1928, the essence of Raman scattering is the interaction between photons and molecular vibration or rotation modes, resulting in a frequency shift of the scattered light, called the Raman shift. This phenomenon provides an important means for molecular structure analysis. Surface-enhanced Raman scattering (SERS) is a phenomenon that uses metal surfaces (such as gold and silver nanoparticles) to enhance Raman signals. In the early 1980s, experiments by Kneipp et al. first discovered that the sensitivity of molecular detection can be significantly improved by enhancing Raman scattering on metal surfaces. The enhancement effect of SERS stems from the enhancement of surface plasmon resonance and local electromagnetic fields, which significantly increases the Raman scattering intensity of molecules near the metal surface and is widely used in biosensing, environmental monitoring and other fields.

[0003] Heavy metal pollution in seawater, especially lead ions (Pb 2+ ), mainly from industrial wastewater discharge, mining, agricultural fertilization and urban sewage. Once lead ions enter the marine environment, they cause serious harm to the marine ecosystem and can affect aquatic organisms and human health through the food chain, leading to a variety of diseases such as nervous system damage and kidney damage. The detection of lead ions faces many challenges, mainly due to the complex background interference in seawater, the need to detect low-concentration lead ions, and their coexistence with other ions or organic matter in seawater. In addition, lead ions lack obvious Raman-active vibrational modes, making it difficult for traditional Raman spectroscopy to directly perform high-sensitivity detection. Although SERS technology has high sensitivity, it also faces certain difficulties in detecting lead ions. Since lead ions themselves do not have obvious typical Raman characteristics, SERS detection usually requires the use of specific metal nanomaterials as an enhanced substrate and relies on the interaction between lead ions and other molecules or substrates. Despite this, SERS still has the advantages of fast response, low detection limit and non-destructiveness, and is suitable for high-sensitivity detection of lead ions in seawater.

[0004] Among common surface-enhanced Raman scattering (SERS) substrate fabrication processes, ultraviolet (UV) lithography has been widely used to prepare various micro- and nano-scale solid-state substrates. UV lithography utilizes ultraviolet light with a shorter wavelength for exposure. Compared to traditional visible light lithography, the shorter wavelength of UV light allows for finer patterns to be etched. This enables UV lithography to precisely transfer patterns at the nanometer scale, meeting the high-resolution requirements of modern SERS substrate fabrication. However, as the size of the mask pattern decreases, the diffraction effect of light becomes more pronounced, making it impossible to accurately replicate the pattern on the mask at the nanometer scale. Femtosecond laser processing is an advanced and rapid technique that allows for intuitive substrate structure fabrication. However, processing accuracy is also limited by factors such as spot size and wavelength, resulting in relatively rough processing at the nanometer scale. Failure to achieve nanoscale processing results in weak local electric field enhancement, insufficient molecular adsorption sites, and uneven nanostructure morphology, all of which can lead to SERS signal attenuation. Therefore, precise nanoscale processing is crucial for obtaining strong and stable SERS signals. Based on the existing conditions, the principle of laser-induced plasma expansion was used to break through the upper limit of process precision, and a new SERS substrate combining multiple processes was developed for sensitive quantitative detection of trace targets.

[0005] Based on the above-mentioned deficiencies in the prior art, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a novel SERS substrate fabricated using the principle of laser-induced plasma expansion and a combination of multiple processes. The present invention incorporates an appropriate concentration of gold nanoparticles into PMMA and casts it into a cylindrical substrate. This substrate is then modified with a femtosecond laser, subjected to secondary crater processing, and gold-plated on the surface to form the final crater substrate with a micro-nanostructure. The present invention utilizes the effective and specific recognition of lead ions with DNAzymes to successfully apply the crater substrate to SERS trace detection of lead ions in seawater.

[0007] To achieve the above object, the technical solution provided by the present invention is:

[0008] A method for preparing a femtosecond laser-induced plasma (SERS) substrate comprises the following steps:

[0009] Gold nanoparticles at an appropriate concentration are doped into PMMA to form a cylindrical substrate, which is then modified with a femtosecond laser, subjected to secondary crater processing, and gold-plated on the surface to form the final crater substrate with a micro-nano structure.

[0010] Specifically:

[0011] Step S1, preparation of 60 nm gold nanoparticles;

[0012] According to the citrate reduction method, boil 75 ml of 2.2 mM sodium citrate solution and add 0.5 ml of 25 mM HAuCl4 solution dropwise. When the color of the solution in the flask remains red, lower the temperature to 90 degrees Celsius and add 0.5 ml of 60 mM sodium citrate solution. After two minutes, add 0.5 ml of 25 mM HAuCl4 solution. Repeat this process 10-12 times. Then, maintain the temperature at 90 degrees Celsius for approximately 30 minutes and allow to cool naturally to room temperature.

[0013] Step S2, preparation of liquid PMMA;

[0014] PMMA powder and ethyl lactate were mixed and stirred at a mass ratio of 1:6 for about 12 hours, and then vacuumed to remove excess bubbles to finally produce a uniform colloidal PMMA.

[0015] Step S3, preparation of PDMS mold;

[0016] Su8-3035 photoresist is spread across the entire silicon wafer using a spin coater. Using a UV photolithography machine, the pattern with the base structure on the film is imprinted onto the silicon substrate under the light-induced crosslinking effect. After drying the silicon substrate, a gel-like PDMS mixed with a crosslinker is poured into it. After removing air bubbles and drying naturally for approximately 4 hours, the structured PDMS is molded into a mold and embedded in a glass culture dish.

[0017] Step S4, preparation of gold nanoparticle-doped PMMA film;

[0018] Transfer 1ml of 60nm gold sol to a centrifuge tube and centrifuge at 5000rpm for 8-10 minutes. Aspirate 950μl of the supernatant and resuspend the remaining portion in 50μl of 50% ethanol. Mix the resuspended gold nanoparticles with PMMA and thoroughly mix using a vibrating mixer. Pour the mixture into a mold and dry it in an oven. Optimize the amount of gold sol added to control the plasma concentration in the film, thereby changing the final measured SERS signal. To optimize the plasma concentration, the amount of gold sol added was controlled to 0.25ml, 0.5ml, 0.75ml, 1ml, and 1.25ml.

[0019] Step S5, preparation of plasma-doped SERS substrate;

[0020] The prepared PMMA film with columnar structure was placed in the femtosecond laser galvanometer processing sample stage, and the laser energy power was set to 20mW and the scanning speed was set to 5mm / s for surface modification;

[0021] The modified film is transferred to a three-dimensional displacement table of femtosecond laser objective machining, a laser energy of 100 mW is set, a single micro column is perforated, a vacuum coating instrument is used to plate gold on the surface of the substrate, and finally a SERS substrate of PMMA doped with crater-shaped gold nanoparticles is prepared;

[0022] In order to optimize the SERS performance of the substrate, the scanning speed is adjusted to 5 mm / s, 10 mm / s, 15 mm / s, 20 mm / s and 25 mW during modification, and the laser energy is adjusted to 80 mW, 100 mW, 120 mW and 140 mW during processing.

[0023] The application also provides a technical scheme of applying the SERS substrate prepared by the preparation method of the femtosecond laser-induced plasma SERS substrate in a microfluidic chip.

[0024] Specifically, the preparation method of the microfluidic chip using the femtosecond laser-induced plasma SERS substrate is as follows:

[0025] An AutoCAD software is used to design a polydimethylsiloxane (PDMS) mold for imprinting patterns on a glass slide covered with photoresist; first, a high-resolution mask aligner is used to process the Su8-3035 photoresist mold on a silicon wafer, and the formation of the pattern is accurately controlled; then, the PDMS prepolymer is mixed with the curing agent at a mass ratio of 10:1, and a vacuum pump is used to remove the bubbles in the mixture to ensure the uniformity of the material and minimize defects; in order to form the cavity structure of the reaction chamber, a stainless steel block is used as a separation tool, and after the degassed PDMS is poured into the mold, it is placed in a 70℃ oven for curing for about 2 hours; after curing, the PDMS layer is taken out and the stainless steel block is carefully peeled off from the reaction chamber, thereby obtaining the predetermined cavity structure; subsequently, two holes are punched in the PDMS layer, which are used as liquid inlet and outlet respectively; next, the PDMS layer and the glass slide are subjected to about 90 seconds of oxygen plasma treatment to enhance the surface affinity, ensuring that the two can be firmly bonded; immediately after the treatment, the PDMS layer and the glass slide are sealed to complete the packaging of the reaction chamber. The PDMS reaction chamber will be used to load the new type of plasma-doped SERS substrate as the core component of the subsequent detection experiment; wherein,

[0026] The chip is composed of a sample inlet area, a mixing channel area, a detection area and a power area, the power area includes a split channel, a plurality of capillary channels and a collection channel; the pump-free microfluidic chip utilizes capillary force to drive fluid, without the need for an external pump system, simplifying the chip structure, reducing cost and complexity; it is easy to operate and integrate, suitable for portable and on-site applications, and can save energy and improve efficiency compared to traditional pump systems; at the same time, due to the reduction of mechanical components and potential failure points, the reliability and stability of the system are improved;

[0027] This method successfully achieved high-precision PDMS mold production and reaction chamber construction through reasonable material selection and process optimization.

[0028] The present invention also provides a technical solution for the application of a femtosecond laser induced plasma SERS substrate microfluidic chip in the detection of lead ions in seawater.

[0029] Specifically:

[0030] Step S6, aptamer binding and modification;

[0031] 4 μl of 10 μM of two single-stranded DNAs were mixed with 72 μl of PBS buffer, incubated at 95°C for approximately 10 minutes, and then cooled to room temperature. 0.8 μl of 5 mM TCEP was added and incubated for approximately 1 hour. The cut substrates were treated using a PLASMA oxygen plasma cleaner, removed and placed in the incubated DNA, immersed in the incubated DNA and incubated in the dark for approximately 12 hours with shaking to allow the double-stranded DNA to fully bind to the novel SERS substrate. The treated substrates were then embedded in a microfluidic chip and sealed, and lead ion solution samples of varying concentrations to be tested were added.

[0032] Step S7, SERS detection of lead ions on the microfluidic chip;

[0033] After aptamer binding and modification in step S6, the chip was placed on a Renishaw Raman test sample stage, and SERS detection was performed on the substrate in the reaction chamber and the results were collected. The spectrometer parameters were set as follows: excitation light source of 633 nm, 50× microscope objective (0.5NA), laser power of 5 mW, integration time of 5 s, and integration number of 1. All Raman spectra were background subtracted and smoothed using Wire 5.5 software, and the average SERS spectrum of 5 or more scans was obtained as the final SERS result.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. A new type of micro-nano substrate was designed, which broke through the upper limit of the original process precision by using the principle of plasma expansion, achieved submicron-level micromachining, and was used for trace gradient detection of lead ions.

[0036] 2. A microfluidic SERS sensor was designed, which can achieve a low detection limit and exceed the national standard while realizing indirect detection of lead ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a flow chart for preparing a femtosecond laser-induced plasma SERS substrate according to the present invention;

[0038] Figure 2 Schematic diagram illustrating the various components of a microfluidic chip using a femtosecond laser-induced plasmon SERS substrate;

[0039] Figure 3 This is a physical picture of the laser-induced plasma SERS substrate of the present invention;

[0040] Figure 4 This is a performance verification diagram of the SERS substrate of the present invention;

[0041] Figure 5 This is a waterfall diagram of the SERS substrate of the present invention based on Rhodamine B detection;

[0042] Figure 6 SERS spectra optimized for doped and undoped SERS substrates and measurement positions;

[0043] Figure 7 is the standard Raman spectrum of ROX molecules;

[0044] Figure 8 This is the concentration gradient diagram of lead ion detection by SERS sensor;

[0045] Figure 9 This is the specific detection diagram of SERS sensor for lead ions. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings.

[0047] The purpose of the present invention is to provide a novel SERS substrate fabricated using the principle of laser-induced plasma expansion and a combination of multiple processes. The present invention incorporates an appropriate concentration of gold nanoparticles into PMMA and casts it into a cylindrical substrate. This substrate is then modified with a femtosecond laser, subjected to secondary crater processing, and gold-plated on the surface to form the final crater substrate with a micro-nanostructure. The present invention utilizes the effective and specific recognition of lead ions with DNAzymes to successfully apply the crater substrate to SERS trace detection of lead ions in seawater.

[0048] To achieve the above object, the technical solution provided by the present invention is:

[0049] like Figure 1 As shown in , a method for preparing a femtosecond laser induced plasma SERS substrate of the present invention comprises the following steps:

[0050] Step S1, preparation of 60 nm gold nanoparticles;

[0051] According to the citrate reduction method, boil 75 ml of 2.2 mM sodium citrate solution and add 0.5 ml of 25 mM HAuCl4 solution dropwise. When the color of the solution in the flask remains red, lower the temperature to 85-90 degrees Celsius and add 0.5 ml of 60 mM sodium citrate solution. After two minutes, add 0.5 ml of 25 mM HAuCl4 solution. Repeat this process 10-12 times. Then, maintain the temperature at 85-90 degrees Celsius and heat for 25-30 minutes, then cool naturally to room temperature.

[0052] Step S2, preparation of liquid PMMA;

[0053] PMMA powder and ethyl lactate were mixed at a mass ratio of 1:6 and stirred for 10-12 hours, and then vacuumed to remove excess bubbles to finally produce a uniform colloidal PMMA.

[0054] Step S3, preparation of PDMS mold;

[0055] A Su8-3035 photoresist is spread across the entire silicon wafer using a spin coater. Using a UV photolithography machine, the pattern with the base structure on the film is imprinted onto the silicon substrate under the light-induced crosslinking effect. After drying the silicon substrate, a gel-like PDMS mixed with a crosslinker is poured into it. After removing air bubbles and drying naturally for 3-4 hours, the structured PDMS is molded into a mold and embedded in a glass culture dish.

[0056] The thickness of the photoresist is 45-50 μm. The photolithography step is to place the silicon wafer after the photoresist is removed on a hot plate and heat it at 95°C for 10-15 minutes. The mask is placed close to the silicon wafer and exposed on a photolithography machine. The photoresist in the exposed area undergoes a UV cross-linking reaction, and the pattern on the mask is transferred to the silicon wafer positive template. The exposed silicon wafer is placed on a hot plate and post-baked at 95°C for 2-4 minutes. The post-baked silicon wafer is placed in a container filled with ethyl lactate and gently shaken to remove the unexposed photoresist. After 3-5 minutes, a few drops of isopropyl alcohol are added to the pattern area. If white flocculent precipitates are produced, it means that the development is not complete. The process is continued until only white flocculent precipitates remain in the pattern area on the developed silicon wafer.

[0057] Step S4, preparation of gold nanoparticle-doped PMMA film;

[0058] Transfer 1ml of 60nm gold nanoparticles to a centrifuge tube and centrifuge at 5000rpm for 8-10 minutes. Aspirate 950μl of the supernatant and resuspend the remaining portion in 50μl of 50% ethanol. Mix the resuspended gold nanoparticles with PMMA and thoroughly mix using a vibrating mixer. Pour the mixture into a mold and dry it in an oven. Optimize the amount of gold sol added to control the plasma concentration in the film, thereby changing the final measured SERS signal. To optimize the plasma concentration, the amount of gold sol added was controlled to 0.25ml, 0.5ml, 0.75ml, 1ml, and 1.25ml.

[0059] Step S5, preparation of plasma-doped SERS substrate;

[0060] The prepared PMMA film with a columnar structure was placed in a femtosecond laser galvanometer processing sample stage, and the laser energy power was set to 20mW and the scanning speed was set to 5mm / s for surface modification. The modified film was transferred to a three-dimensional translation stage processed by a femtosecond laser objective lens, and the laser energy power was set to 100mW to punch holes in individual micro-pillars, finally making a crater-shaped gold nanoparticle-doped PMMA SERS substrate.

[0061] The specific steps are shown in the diagram below: Figure 1 As shown in the actual picture Figure 3 As shown, Figure 3 The size of a single crater substrate prepared in A is about 60 μm. Figure 3 B shows that the internal microstructure reaches the nanoscale due to the plasma expansion effect; Figure 4 、 5 , 6 are all performance verifications of the substrate, Figure 4 A Raman reporter molecule RB was used at a concentration of 10 -10 The signal can still be detected at M, which proves the high sensitivity of the substrate. Figure 5 The waterfall chart is to select 10 -6 M RB was used as a target to verify the uniformity of the substrate. Figure 6 Using another reporter molecule MGITC as a target, the different SERS effects of doping and undoping and different detection positions were verified.

[0062] The present invention also provides a technical solution for applying a SERS substrate prepared by a method for preparing a femtosecond laser induced plasma SERS substrate in a microfluidic chip.

[0063] Specifically, the preparation method of the microfluidic chip using a femtosecond laser-induced plasma SERS substrate is as follows:

[0064] A polydimethylsiloxane (PDMS) mold for imprinting patterns on a photoresist-covered glass slide was designed using AutoCAD software. First, a Su8-3035 photoresist mold was processed on a silicon wafer using a high-resolution mask aligner to precisely control pattern formation. The PDMS prepolymer and curing agent were then mixed in a 10:1 mass ratio, and a vacuum pump was used to remove bubbles from the mixture to ensure material uniformity and minimize defects. To form the cavity structure of the reaction chamber, a stainless steel block was used as an isolation tool, and After pouring the degassed PDMS into the mold, it is placed in a 70°C oven to cure for about 2 hours; after curing, the PDMS layer is removed and the stainless steel block is carefully peeled off from the reaction chamber to obtain the predetermined cavity structure; then, two holes are punched in the PDMS layer, which are used as the liquid inlet and outlet respectively; next, the PDMS layer and the glass slide are treated with oxygen plasma for about 90 seconds to enhance their surface affinity and ensure that the two can be firmly bonded; immediately after treatment, the PDMS layer is sealed with the glass slide to complete the packaging of the reaction chamber. The PDMS reaction chamber will be used to load the new plasma-doped SERS substrate as the core component of the subsequent detection experiment; among them,

[0065] like Figure 2 As shown in FIG, the chip comprises an injection area, a mixing channel area, a detection area, and a power area. The power area includes a diversion channel, multiple capillary channels, and a collection channel. The pump-free microfluidic chip utilizes capillary force to drive fluids, eliminating the need for an external pump system. This simplifies the chip structure and reduces cost and complexity. It is easy to operate and integrate, suitable for portable and on-site applications, and can save energy and improve efficiency compared to traditional pump systems. At the same time, the system's reliability and stability are improved by reducing mechanical components and potential failure points.

[0066] This method successfully achieved high-precision PDMS mold production and reaction chamber construction through reasonable material selection and process optimization.

[0067] The present invention also provides a technical solution for the application of a femtosecond laser induced plasma SERS substrate microfluidic chip in the detection of lead ions in seawater.

[0068] Specifically:

[0069] Step S6, aptamer binding and modification;

[0070] 4 μl of 10 μM of two single-stranded DNAs were mixed with 72 μl of PBS buffer, incubated at 95°C for approximately 10 minutes, and then cooled to room temperature. 0.8 μl of 5 mM TCEP was added and incubated for approximately 1 hour. The cut substrates were treated using a PLASMA oxygen plasma cleaner, removed and placed in the incubated DNA, immersed in the incubated DNA and incubated in the dark for approximately 12 hours with shaking to allow the double-stranded DNA to fully bind to the novel SERS substrate. The treated substrates were then embedded in a microfluidic chip and sealed, and lead ion solution samples of varying concentrations to be tested were added.

[0071] Step S7, SERS detection of lead ions on the microfluidic chip;

[0072] After aptamer binding and modification in step S6, the chip was placed on a Renishaw Raman test sample stage, and SERS detection was performed on the substrate in the reaction chamber and the results were collected. The spectrometer parameters were set as follows: excitation light source of 633 nm, 50× microscope objective (0.5NA), laser power of 5 mW, integration time of 5 s, and integration number of 1. All Raman spectra were background subtracted and smoothed using Wire 5.5 software, and the average SERS spectrum of 5 or more scans was obtained as the final SERS result.

[0073] Figure 7 The Raman standard peak of the Raman reporter molecule ROX used to detect lead ions is selected at a wavelength of 1499 cm -1 The peak at is used as a marker to detect the concentration gradient of lead ions. Figure 8 Shown in 10 -12 The signal can still be detected in the lead ion solution of M, exceeding the national standard; Figure 9 The specificity of the SERS microfluidic sensor to lead ions is demonstrated.

[0074] 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 shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a femtosecond laser-induced plasma (SERS) substrate, characterized in that: Gold nanoparticles are doped into PMMA to form a cylindrical substrate, which is then modified by femtosecond laser, subjected to secondary crater processing, and gold-plated on the surface to form the final crater substrate with a micro-nano structure. The specific steps include the following: Step S1, preparation of gold nanoparticles; According to the citrate reduction method, the sodium citrate solution is boiled and the HAuCl4 solution is added dropwise. When the color of the solution in the flask remains red, the temperature is lowered, and the sodium citrate solution is added followed by the HAuCl4 solution. This operation is repeated 10-12 times. The solution is then allowed to cool naturally to room temperature. Step S2, preparation of liquid PMMA; PMMA powder and ethyl lactate were mixed and stirred at a mass ratio of 1:6 for 12 h, and then vacuumed to remove excess bubbles to finally produce a uniform colloidal PMMA. Step S3, preparation of PDMS mold; The Su8-3035 photoresist is spread on the entire silicon wafer by a spin coater, and the pattern with the base structure on the film is engraved on the silicon substrate under the light-induced cross-linking effect of the UV photolithography machine; After drying the silicon substrate, pour the colloidal PDMS mixed with a cross-linking agent into it, remove the bubbles and dry it naturally, and then make a mold of the PDMS with a columnar structure and embed it into a glass culture dish; Step S4, preparation of gold nanoparticle-doped PMMA film; Place the gold sol containing gold nanoparticles into a centrifuge tube, centrifuge at 5000 rpm for 8-10 minutes, remove the supernatant, and resuspend the remaining portion in alcohol; Mix the resuspended gold nanoparticles with PMMA, use a vibrating mixer to mix thoroughly, drop the mixed liquid into the mold and place it in an oven to dry; By optimizing the amount of gold sol added, the concentration of plasma in the film is controlled, thereby changing the final measured SERS signal; Step S5, preparation of plasma-doped SERS substrate; The prepared PMMA film with columnar structure was placed in a femtosecond laser galvanometer processing sample stage, and the laser energy power was set to 20 mW and the scanning speed was set to one of 5 mm / s, 10 mm / s, 15 mm / s, 20 mm / s, and 25 mm / s for surface modification; The modified film was transferred to a three-dimensional translation stage processed by a femtosecond laser objective lens. The laser energy power was set to one of 80mW, 100mW, 120mW, and 140mW, and a single microcolumn was punched to finally produce a crater-shaped SERS substrate of gold nanoparticles doped with PMMA.

2. The method for preparing a femtosecond laser induced plasma (SERS) substrate according to claim 1, wherein: Step S1 specifically comprises: according to the citrate reduction method, boiling 75 ml of a 2.2 mM sodium citrate solution, and adding dropwise 0.5 ml of a 25 mM HAuCl4 solution; when the color of the solution in the flask remains red, lowering the temperature to 90 degrees Celsius, adding 0.5 ml of a 60 mM sodium citrate solution, and then adding 0.5 ml of a 25 mM HAuCl4 solution two minutes later, repeating the operation 10-12 times; then maintaining the temperature at 90 degrees Celsius for 30 minutes, and naturally cooling to room temperature.

3. Use of a SERS substrate prepared by the method for preparing a femtosecond laser induced plasma SERS substrate according to claim 1 or 2 in a microfluidic chip.

4. A microfluidic chip using a femtosecond laser-induced plasma (SERS) substrate according to claim 3, characterized in that: The preparation method comprises the following steps: The PDMS mold was designed using AutoCAD. A Su8 photoresist mold was first fabricated on a silicon wafer using a high-resolution mask aligner. The PDMS prepolymer and curing agent were mixed in a 10:1 ratio, degassed, poured into the mold, and cured. After solidification, the mold is removed, the stainless steel block is peeled off to form a cavity structure, and holes are punched as liquid inlets and outlets; The bonding between PDMS and the glass slide was enhanced by oxygen plasma treatment to complete the reaction chamber encapsulation; This PDMS microfluidic chip is used to load the SERS substrate and is used in subsequent experiments.

5. The microfluidic chip using a femtosecond laser induced plasma (SERS) substrate according to claim 4, characterized in that: Specifically, A polydimethylsiloxane mold for imprinting patterns on a photoresist-covered glass slide was designed using AutoCAD software; First, a Su8-3035 photoresist mold is processed on a silicon wafer using a high-resolution mask aligner to precisely control the formation of the pattern; Then, the PDMS prepolymer and curing agent were mixed in a mass ratio of 10:1, and a vacuum pump was used to remove bubbles from the mixture to ensure material uniformity and minimize defects; In order to form the cavity structure of the reaction chamber, a stainless steel block was used as an isolation tool, and after pouring the degassed PDMS into the mold, it was placed in an oven at 70 °C for 2 h to cure; After curing, the PDMS layer was removed and the stainless steel block was peeled off from the reaction chamber to obtain the predetermined cavity structure; Subsequently, two holes were punched in the PDMS layer to serve as the liquid inlet and outlet, respectively; Next, the PDMS layer and the glass slide were treated with oxygen plasma for 90 seconds to enhance their surface affinity and ensure a strong bond between the two; Immediately after treatment, the PDMS layer was sealed to the glass slide to complete the encapsulation of the reaction chamber.

6. The microfluidic chip using a femtosecond laser induced plasma (SERS) substrate according to claim 5, characterized in that: The microfluidic chip includes an injection area, a mixing channel area, a detection area, and a power area. The power area includes a diversion channel, multiple capillary channels, and a collection channel. The pump-free microfluidic chip uses capillary force to drive fluid through capillary force.

7. Use of a microfluidic chip comprising a femtosecond laser-induced plasma (SERS) substrate as claimed in any one of claims 4 to 6 in detecting lead ions in seawater.

8. Use of the microfluidic chip based on the femtosecond laser induced plasma (SERS) substrate according to claim 7 in detection of lead ions in seawater, characterized in that: The following steps are involved: Step S6, aptamer binding and modification; The double-stranded DNA is incubated with the treated substrate and embedded in a microfluidic chip for sealing; lead ion solutions of different concentrations are added and tested; Step S7, SERS detection of lead ions on the microfluidic chip; The microfluidic chip loaded with the substrate was transferred to a Raman spectrometer for SERS detection.

9. Use of the microfluidic chip based on the femtosecond laser induced plasma (SERS) substrate according to claim 8 in detection of lead ions in seawater, characterized in that: The step S6 is specifically as follows: 4 μl of 10 μM of two single-stranded DNAs were mixed with 72 μl of PBS buffer, incubated in a 95°C water bath for 10 min, and then cooled to room temperature; Add 0.8 μl of 5 mM TCEP and incubate for 1 hour. Use a PLASMA oxygen plasma cleaner to treat the cut substrate. After taking it out, put it into the incubated DNA and soak it in the dark with shaking for 12 hours to allow the double-stranded DNA to fully bind to the SERS substrate. The treated substrate is embedded in a microfluidic chip and sealed, and lead ion solution samples with different concentrations to be tested are added.

10. Use of the microfluidic chip based on the femtosecond laser induced plasma (SERS) substrate according to claim 8 in detection of lead ions in seawater, characterized in that: The step S7 is specifically as follows: After aptamer binding and modification in step S6, the chip was placed on a Renishaw Raman test sample stage, and SERS detection was performed on the substrate in the reaction chamber and the results were collected. The spectrometer parameters were set as follows: excitation light source of 633nm, 50× microscope objective, laser power of 5mW, integration time of 5s, and integration number of 1. All Raman spectra were background subtracted and smoothed using Wire 5.5 software, and the average SERS spectrum of more than 5 scans was obtained as the final SERS result.

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