Preparation method and application of femtosecond laser induced plasma SERS (Surface Enhanced Raman Scattering) substrate

Abstract

The invention 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 application of the plasma SERS substrate micro-fluidic chip in trace detection of lead ions in seawater. According to the method, a polymethyl methacrylate (PMMA) material doped with gold nanoparticles is adopted, a cylindrical template is prepared on a silicon wafer through an ultraviolet lithography technology, and the gold-doped PMMA is poured into the template to prepare a cylindrical structure. Then, the outer surface of the substrate is roughened through femtosecond laser modification, and finally, the cylindrical structure is machined into the micro-nano structure with the crater morphology through the femtosecond laser machining technology. According to the invention, the principle of laser-induced plasma expansion is utilized, the precision upper limit of the original micron-level process is broken through, the structure regulation of nano-level precision is realized, the SERS signal of the substrate is greatly improved, and the detection sensitivity of lead ions is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of micro-nano sensing and SERS detection, is aimed at detecting lead ions in seawater, and relates to a method and application of preparing a SERS substrate by 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 CV Raman in 1928, the essence of Raman scattering is the interaction between photons and molecular vibration or rotation modes, which leads to a frequency shift of scattered light, called 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 originates 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. They 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 vibration 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 the common surface enhanced Raman scattering substrate preparation processes, ultraviolet lithography has been widely used in the preparation of various micro-nano solid-state substrates. Ultraviolet lithography uses ultraviolet light with a shorter wavelength for exposure. Compared with traditional visible light lithography, the wavelength of ultraviolet light is shorter and can etch finer patterns. This allows ultraviolet lithography to perform precise pattern transfer at the nanometer level, meeting the high-resolution requirements of modern SERS substrate manufacturing. However, as the size of the mask pattern decreases, the diffraction effect of light becomes more obvious, resulting in the inability to accurately replicate the pattern on the mask at the nanometer scale. Femtosecond laser processing technology is an advanced and fast processing technology that can intuitively realize substrate structure processing. However, the processing accuracy is also limited by the spot size, wavelength, etc., and the processing is often relatively rough at the nanometer scale. If the process does not reach the nanometer level, the local electric field enhancement will not be obvious, the molecular adsorption sites will be insufficient, and the nanostructure morphology will be uneven, all of which will lead to the attenuation of the SERS signal. Therefore, fine processing at the nanometer level is crucial to obtain 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 accuracy, 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 new SERS substrate prepared by using the principle of laser-induced plasma expansion and combining multiple processes. The present invention dopes gold nanoparticles of appropriate concentration into PMMA to form a cylindrical substrate, and then forms a final crater substrate with a micro-nano structure through femtosecond laser modification, secondary processing of the crater, and gold plating on the surface. The present invention utilizes the effective and specific recognition of lead ions and DNAzyme to successfully apply the crater substrate to the SERS trace detection of lead ions in seawater;

[0007] To achieve the above purpose, 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] The appropriate concentration of gold nanoparticles was doped into PMMA to form a cylindrical substrate, and then the crater substrate with micro-nano structure was formed through femtosecond laser modification, secondary crater processing and surface gold plating.

[0010] Specifically:

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

[0012] According to the citrate reduction method, 75 ml of 2.2 mM sodium citrate solution was boiled, and 0.5 ml of 25 mM HAuCl4 solution was added dropwise; when the color of the solution in the flask remained red, the temperature was lowered to 90 degrees, and 0.5 ml of 60 mM sodium citrate solution was added. After two minutes, 0.5 ml of 25 mM HAuCl4 solution was added, and the operation was repeated 10-12 times; then the temperature was maintained at 90 degrees and heated for about 30 minutes, and then cooled 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 the excess bubbles were removed by vacuum to finally prepare a uniform colloidal PMMA;

[0015] Step S3, preparation of PDMS mold;

[0016] The Su8-3035 photoresist is spread on the entire silicon wafer by a coating machine, and the pattern with the base structure on the film plate is engraved on the silicon substrate under the light-induced cross-linking effect of the UV photolithography machine. After the silicon substrate is dried, the gelatinous PDMS mixed with the cross-linking agent is poured into it, and after removing the bubbles, it is naturally dried for about 4 hours. The PDMS with the structure is made into a mold and embedded in a glass culture dish;

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

[0018] Take 1ml of 60nm gold sol into a centrifuge tube, centrifuge at 5000rpm for 8-10min, remove 950μl of supernatant, and resuspend the remaining part with 50μl of 50% alcohol. Mix the resuspended gold nanoparticles with PMMA, use a vibrating mixer to mix thoroughly, drop the mixed liquid into the mold and put it in an oven to dry; control the concentration of plasma in the film by optimizing the amount of gold sol added, thereby changing the final measured SERS signal. In order to optimize the plasma concentration, the amount of gold sol added is controlled to 0.25ml, 0.5ml, 0.75ml, 1ml, 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 20 mW and the scanning speed was set to 5 mm / s for surface modification;

[0021] The modified film was transferred to a three-dimensional translation stage processed by a femtosecond laser objective lens, the laser energy was set to 100 mW, a single microcolumn was punched, and gold was plated on the substrate surface using a vacuum coating apparatus, and finally a crater-shaped gold nanoparticle-doped PMMA SERS substrate was produced;

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

[0023] 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.

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

[0025] 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 the pattern formation. Then, the PDMS prepolymer was mixed with a curing agent 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, place it in a 70°C oven to cure for about 2 hours; after curing, remove the PDMS layer and carefully peel off the stainless steel block from the reaction chamber to obtain the predetermined cavity structure; then, punch two holes 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 subsequent detection experiments; among them,

[0026] The chip consists of an injection area, a mixing channel area, a detection area, and a power area. The power area includes a shunt channel, multiple capillary channels, and a collection channel. The pump-free microfluidic chip uses capillary force to drive the fluid, without the need for an external pump system, which simplifies the chip structure and reduces cost and complexity. It is easy to operate and integrate, suitable for portable and field applications, and can save energy and improve efficiency compared to traditional pump systems. At the same time, the reliability and stability of the system are improved due to the reduction of mechanical components and potential failure points.

[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, binding and modification of aptamers;

[0031] 4 μl, 10 μM of two single-stranded DNAs were mixed with 72 μl of PBS buffer, and cooled to room temperature after being placed in a 95-degree water bath for about 10 minutes; 0.8 μl, 5 mM TCEP was added and incubated for about 1 hour; the cut substrates were treated with a PLASMA oxygen plasma cleaner, and then taken out and placed in the incubated DNA for soaking and shaking incubation for about 12 hours in the dark, so that the double-stranded DNA was fully combined with the new SERS substrate; the treated substrate was embedded in a microfluidic chip and sealed, and lead ion solution samples of different concentrations to be tested were added;

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

[0033] After the aptamer binding and modification in step S6, the chip was placed on the Renishaw Raman test sample stage, and the substrate in the reaction chamber was subjected to SERS detection and the results were collected; the parameters of the spectrometer were set as follows: the excitation light source was 633 nm, the microscope objective lens was 50× (0.5NA), the laser power was 5 mW, the integration time was 5 s, and the number of integrations was 1; all Raman spectra were background subtracted and smoothed using the Wire 5.5 software, and then 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 used the plasma expansion principle to break through the upper limit of the original process accuracy, achieved submicron-level micromachining, and used the substrate 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 of the preparation of a femtosecond laser induced plasma SERS substrate according to the present invention;

[0038] Figure 2 A schematic diagram illustrating the various parts 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 It is a waterfall diagram of the SERS substrate of the present invention based on the detection of Rhodamine B;

[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 molecule;

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

[0045] Fig. 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 in conjunction with the accompanying drawings.

[0047] The purpose of the present invention is to provide a new SERS substrate prepared by using the principle of laser-induced plasma expansion and combining multiple processes. The present invention dopes gold nanoparticles of appropriate concentration into PMMA to form a cylindrical substrate, and then forms a final crater substrate with a micro-nano structure through femtosecond laser modification, secondary processing of the crater, and gold plating on the surface. The present invention utilizes the effective and specific recognition of lead ions and DNAzyme to successfully apply the crater substrate to the SERS trace detection of lead ions in seawater;

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

[0049] like Figure 1 As shown in the figure, 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, 75 ml of 2.2 mM sodium citrate solution was boiled, and 0.5 ml of 25 mM HAuCl4 solution was added dropwise; when the color of the solution in the flask remained red, the temperature was lowered to 85-90 degrees, 0.5 ml of 60 mM sodium citrate solution was added, and 0.5 ml of 25 mM HAuCl4 solution was added after two minutes, and the operation was repeated 10-12 times; then the temperature was maintained at 85-90 degrees and heated for 25-30 minutes, and then cooled naturally to room temperature;

[0052] Step S2, preparation of liquid PMMA;

[0053] PMMA powder and ethyl lactate were mixed and stirred at a mass ratio of 1:6 for 10-12 hours, and excess bubbles were removed by vacuuming to finally prepare a uniform colloidal PMMA;

[0054] Step S3, preparation of PDMS mold;

[0055] The Su8-3035 photoresist is spread on the entire silicon wafer by a coating machine, and the pattern with the base structure on the film plate is engraved on the silicon substrate under the light-induced cross-linking effect of the UV photolithography machine. After the silicon substrate is dried, the gelatinous PDMS mixed with the cross-linking agent is poured into it, and after removing the bubbles, it is naturally dried for 3-4 hours, and the PDMS with the structure is made 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 thrown on a heating plate, heat it at 95°C for 10-15 minutes, place the mask close to the top of the silicon wafer, and expose it on a photolithography machine. The photoresist in the exposed area undergoes an ultraviolet cross-linking reaction, and the pattern on the mask is transferred to the positive template of the silicon wafer. The exposed silicon wafer is placed on a heating 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. This step is to remove the unexposed photoresist. After 3-5 minutes, drop a few drops of isopropyl alcohol on the pattern area. If white flocculent precipitates are produced, it means that the development is not clean. The pattern area on the developed silicon wafer has only white flocculent precipitates.

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

[0058] Take 1ml of 60nm gold nanoparticles into a centrifuge tube, centrifuge at 5000rpm for 8-10min, remove 950μl of supernatant, and resuspend the remaining part with 50μl of 50% alcohol. Mix the resuspended gold nanoparticles with PMMA, use a vibrating mixer to mix thoroughly, drop the mixed liquid into the mold and put it in an oven to dry; control the concentration of plasma in the film by optimizing the amount of gold sol added, thereby changing the final measured SERS signal. In order to optimize the plasma concentration, the amount of gold sol added is controlled to 0.25ml, 0.5ml, 0.75ml, 1ml, 1.25ml;

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

[0060] 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; the modified film was transferred to the three-dimensional translation stage processed by the femtosecond laser objective lens, and the laser energy power was set to 100mW to perform a hole punching operation on a single micro-column, and finally a crater-shaped gold nanoparticle-doped PMMA SERS substrate was prepared;

[0061] The specific steps are shown in the figure 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 has reached the nanoscale due to the plasma expansion effect; Figure 4 , 5 , 6 are the performance verification 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 the 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 the pattern formation. Then, the PDMS prepolymer was mixed with a curing agent 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, place it in a 70°C oven to cure for about 2 hours; after curing, remove the PDMS layer and carefully peel off the stainless steel block from the reaction chamber to obtain the predetermined cavity structure; then, punch two holes 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 subsequent detection experiments; among them,

[0065] like Figure 2 As shown, the chip consists of a sample injection area, a mixing channel area, a detection area, and a power area. The power area includes a shunt channel, multiple capillary channels, and a collection channel. The pump-free microfluidic chip using capillary force drives the fluid through capillary force, without the need for an external pump system, thus simplifying the chip structure and reducing costs 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 reliability and stability of the system are improved due to the reduction of 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, binding and modification of aptamers;

[0070] 4 μl, 10 μM of two single-stranded DNAs were mixed with 72 μl of PBS buffer, and cooled to room temperature after being placed in a 95-degree water bath for about 10 minutes; 0.8 μl, 5 mM TCEP was added and incubated for about 1 hour; the cut substrates were treated with a PLASMA oxygen plasma cleaner, and then taken out and placed in the incubated DNA for soaking and shaking incubation for about 12 hours in the dark, so that the double-stranded DNA was fully combined with the new SERS substrate; the treated substrate was embedded in a microfluidic chip and sealed, and lead ion solution samples of different concentrations to be tested were added;

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

[0072] After the aptamer binding and modification in step S6, the chip was placed on the Renishaw Raman test sample stage, and the substrate in the reaction chamber was subjected to SERS detection and the results were collected; the parameters of the spectrometer were set as follows: the excitation light source was 633 nm, the microscope objective lens was 50× (0.5NA), the laser power was 5 mW, the integration time was 5 s, and the number of integrations was 1; all Raman spectra were background subtracted and smoothed by Wire 5.5 software, and then 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 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; Fig. 9 The specificity of the SERS microfluidic sensor to lead ions is demonstrated.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a femtosecond laser induced plasma SERS substrate, characterized in that: Gold nanoparticles are doped into PMMA to form a cylindrical substrate, and then the crater substrate with a micro-nano structure is formed by femtosecond laser modification, secondary crater processing and surface gold plating. The specific steps are as follows: 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, the sodium citrate solution is added, and then the HAuCl4 solution is added, and the operation is repeated 10-12 times; and the solution is naturally cooled 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 about 12 hours, and the excess bubbles were removed by vacuum to finally prepare a uniform colloidal PMMA; Step S3, preparation of PDMS mold; The Su8-3035 photoresist is spread on the entire silicon wafer by a coating machine, and the pattern with the base structure on the film plate is engraved on the silicon substrate under the light-induced cross-linking action of the ultraviolet photolithography machine; After drying the silicon substrate, pour the gelatinous PDMS mixed with a cross-linking agent into it, remove the bubbles and dry it naturally, and 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 min, remove the supernatant, and resuspend the remaining portion with alcohol; Mix the resuspended gold nanoparticles with PMMA, use a vibrating mixer to mix thoroughly, drop the mixed liquid into a mold and put 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 the three-dimensional translation stage processed by the femtosecond laser objective lens, and the laser energy power was set to 80mW. One of 100 mW, 120 mW, and 140 mW was used to perform a hole punching operation on a single microcolumn, and finally a crater-shaped gold nanoparticle-doped PMMA SERS substrate was produced.

2. The method for preparing a femtosecond laser induced plasma (SERS) substrate according to claim 1, characterized in that: The step S1 is specifically as follows: according to the citrate reduction method, 75 ml of 2.2 mM sodium citrate solution is boiled, and 0.5 ml of 25 mM HAuCl4 solution is added dropwise; when the color of the solution in the flask remains red, the temperature is lowered to 90 degrees, 0.5 ml of 60 mM sodium citrate solution is added, and 0.5 ml of 25 mM HAuCl4 solution is added after two minutes, and the operation is repeated 10-12 times; then the temperature is maintained at 90 degrees for about 30 minutes, and then naturally cooled 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. The 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. First, a Su8 photoresist mold was processed on a silicon wafer using a high-resolution mask aligner. The PDMS prepolymer and curing agent were mixed at a ratio of 10:1, degassed, poured into the mold, and cured. After solidification, the mold is taken out, 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 applied 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 (PDMS) 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 the uniformity of the material 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 curing for about 2 h; After curing, the PDMS layer is removed and the stainless steel block is peeled off from the reaction chamber to obtain a predetermined cavity structure; Subsequently, two holes were punched on 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 that the two could be firmly bonded; Immediately after treatment, the PDMS layer was sealed with a 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 comprises a sample injection area, a mixing channel area, a detection area, and a power area, wherein the power area comprises a shunt channel, a plurality of capillary channels, and a collection channel; the pump-free microfluidic chip utilizing capillary force drives fluid by capillary force.

7. Use of a microfluidic chip based on a femtosecond laser induced plasma (SERS) substrate as claimed in any one of claims 4 to 6 in the detection of lead ions in seawater.

8. The use of the microfluidic chip based on the femtosecond laser induced plasma (SERS) substrate according to claim 7 in the detection of lead ions in seawater, characterized in that: The following steps are involved: Step S6, binding and modification of aptamers; Incubate the double-stranded DNA with the treated substrate and embed it into a microfluidic chip for sealing; add lead ion solutions of different concentrations and wait for detection; Step S7, SERS detection of lead ions on the microfluidic chip; The microfluidic chip loaded with the substrate was transferred to the Raman spectrometer for SERS detection.

9. The use of the microfluidic chip based on the femtosecond laser induced plasma (SERS) substrate according to claim 8 in the 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 about 10 min, and then cooled to room temperature; Add 0.8 μl of 5 mM TCEP and incubate for about 1 hour; use a PLASMA oxygen plasma cleaner to treat the cut substrate, take it out and put it into the incubated DNA for soaking and shaking in the dark for about 12 hours to allow the double-stranded DNA to fully bind to the new 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. The use of the microfluidic chip based on the femtosecond laser induced plasma (SERS) substrate according to claim 8 in the detection of lead ions in seawater, characterized in that: The step S7 is specifically as follows: After the aptamer binding and modification in step S6, the chip was placed on the Renishaw Raman test sample stage, and the substrate in the reaction chamber was subjected to SERS detection and the results were collected; the parameters of the spectrometer were set as follows: the excitation light source was 633 nm, the microscope objective lens was 50× (0.5NA), the laser power was 5 mW, the integration time was 5 s, and the number of integrations was 1; all Raman spectra were background subtracted and smoothed using the Wire 5.5 software, and then the average SERS spectrum of 5 or more scans was obtained as the final SERS result.

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