Antigen-coupled PVDF-PEI membrane type SERS (Surface Enhanced Raman Scattering) sensor and application thereof

Abstract

The invention discloses an antigen-coupled PVDF-PEI membrane type SERS sensor constructed based on a surface enhanced Raman scattering (SERS) technology, the antigen-coupled PVDF-PEI membrane type SERS sensor comprises two parts of a specific immune probe and an antigen-coupled PVDF-PEI composite membrane, and the preparation of the antigen-coupled PVDF-PEI membrane type SERS sensor comprises the following steps: synthesizing Au (at) 4-MBA (at) Ag NPs with a core-shell structure through gold nanoparticles (AuNPs), and coupling an METH antibody to obtain an SERS immune probe; meanwhile, a PVDF film is subjected to pretreatment, surface PEI modification and tannic acid modification to prepare a PVDF-PEI composite film, and then the PVDF-PEI composite film is coupled with an METH antigen (METH-BSA) to form the detection substrate. The prepared antigen-coupled PVDF-PEI membrane type SERS sensor can be used for ultra-trace detection of methylamphetamine (METH) and derivatives thereof in a liquid sample, has the advantages of high sensitivity, good anti-interference capability and the like, and has very huge popularization and application values.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to an antigen-coupled PVDF-PEI film-type SERS sensor constructed based on surface-enhanced Raman scattering (SERS) technology, which can be used for ultra-trace detection of methamphetamine (METH) and its derivatives. Background Technology

[0002] In recent years, the proliferation of illicit drugs has increasingly become a global concern. Methamphetamine and its derivatives, due to their ease of preparation and low cost, have become one of the most widely abused central nervous system stimulants worldwide. The abuse of these drugs not only seriously damages individual health but also poses a severe threat to social stability and economic development. Studies have shown that methamphetamine and its metabolites enter water bodies through wastewater, industrial emissions, and domestic sewage, becoming a new type of environmental pollutant. Especially in areas with high abuse rates, the concentration of methamphetamine in sewage increases significantly, posing a potential threat to ecosystems and water resource security. Therefore, seeking a rapid and reliable method for detecting methamphetamine in environmental sewage is crucial for assessing pollution levels, developing pollution control measures, and tracking drug use.

[0003] Currently, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and high-performance liquid chromatography (HPLC) exhibit good sensitivity and specificity in detecting trace amounts of ketamine, methylenedioxymethamphetamine, and methamphetamine derivatives. However, their limitations stem from high instrument costs, cumbersome sample pretreatment, and the high level of expertise required of operators, making it difficult to meet the rapid and convenient on-site testing needs of public security departments. In addition, paper-based rapid screening tools based on immunolateral flow chromatography, such as colloidal gold test strips, have been developed in recent years. These strips are easy to operate and low in cost, and are widely used in on-site testing. However, their results are easily affected by the complex buffer systems of the test liquids, leading to cross-reaction interference, and they also suffer from low sensitivity and the inability to quantify the results.

[0004] Surface-enhanced Raman scattering (SERS), as an emerging spectroscopic detection technique with ultra-high sensitivity and molecular specificity, has shown great application potential in the field of trace substance analysis. The basic principle of SERS is to significantly enhance the Raman scattering signal intensity of target molecules through the localized surface plasmon resonance effect on the surface of nanostructured metals. In recent years, research on SERS technology in pollutant analysis has gradually increased, particularly showing broad application prospects in the detection of trace drugs, pesticides, and heavy metals. However, the application of SERS technology in complex matrices still faces some challenges, such as matrix effects causing signal interference and the stability of nanomaterial substrates. Furthermore, the development of portable devices and rapid on-site analysis remain current research hotspots and difficulties. Summary of the Invention

[0005] The purpose of this invention is to provide an antigen-coupled PVDF-PEI membrane SERS sensor and to use it for ultra-trace detection of methamphetamine (METH) and its derivatives.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: An antigen-coupled PVDF-PEI membrane-type SERS sensor constructed based on surface-enhanced Raman scattering (SERS) technology comprises two parts: a specific immune probe and an antigen-coupled PVDF-PEI composite membrane. Its preparation method includes the following steps: 1) Preparation of specific immune probes a) Synthesis of gold nanoparticles (AuNPs): Chloroauric acid solution was added to water and stirred vigorously and heated. When the solution boiled, sodium citrate solution, a reducing agent, was quickly added. The mixture was heated and stirred for 15 minutes and then cooled to room temperature to obtain AuNPs solution. b) Synthesis of Au@Raman signal molecule solution: Add Raman signal molecule solution to the obtained AuNPs solution, incubate, centrifuge to remove excess Raman signal molecules, and then resuspend in ultrapure water to obtain Au@Raman signal molecule solution; c) Synthesis of Au@Raman signal molecule@Ag solution: Sodium hydroxide solution, sodium citrate solution, ascorbic acid solution and silver nitrate solution were added sequentially to the obtained Au@Raman signal molecule solution. After stirring and reacting thoroughly, the supernatant was removed by centrifugation, and the precipitate was resuspended in water to obtain Au@Raman signal molecule@Ag solution. d) Synthesis of specific nanoprobe solution: After adding dithiobissuccinimide propionate (DSP) solution to the obtained Au@Raman signal molecule@Ag solution and reacting, METH antibody solution with biological specific recognition function was added, and then incubated at 37°C to functionalize and couple Au@Raman signal molecule@Ag with METH antibody. After resuspending and blocking with bovine serum albumin (BSA), specific nanoprobe solution was obtained. 2) Preparation of antigen-conjugated PVDF-PEI composite membrane e) Pretreatment of polyvinylidene fluoride (PVDF) membrane: After cutting the PVDF membrane to the required size, immerse it in isopropanol, and then rinse it repeatedly with running water to obtain the pretreated PVDF membrane. f) Preparation of PVDF-PEI membrane: The pretreated PVDF membrane was immersed in a mixed solution of polyethyleneimine (PEI) and 3-chloropropyltrimethoxysilane (CTS) and stirred at room temperature to obtain PVDF-PEI membrane; g) Modification of PVDF-PEI membrane: The prepared PVDF-PEI membrane was immersed in Tris-HCl buffer containing tannic acid (TA) at room temperature, and then dried to obtain the modified PVDF-PEI composite membrane. h) Antigen conjugation on PVDF-PEI membrane: METH antigen is covalently fixed onto PVDF-PEI membrane via amide bonds to obtain antigen-conjugated PVDF-PEI composite membrane.

[0007] Further, the concentration of the chloroauric acid solution in step a) is 1 wt%.

[0008] Further, the concentration of the sodium citrate solution in step a) is 1 wt%.

[0009] Furthermore, the volume ratio of chloroauric acid solution, water and sodium citrate solution used in step a) is 1:99:1.5.

[0010] Furthermore, the concentration of the Raman signal molecule solution in step b) is 1 mM, and the Raman signal molecule used is 4-mercaptobenzoic acid (MBA).

[0011] Furthermore, in step b), the amount of Raman signal molecule solution added is calculated as 1 μL per milliliter of AuNPs solution.

[0012] Furthermore, the incubation time described in step b) is 0.5 to 1 hour.

[0013] Further, in step c), 10 μL of 0.1 mM sodium hydroxide solution, 100 μL of 1 wt% sodium citrate solution, 1 mL of 10 mM ascorbic acid solution and 1 mL of 1 mM silver nitrate solution are added to every 10 mL of Au@Raman signal molecule solution.

[0014] Furthermore, the stirring reaction time in step c) is 15 min.

[0015] Further, the concentration of the dithiobissuccinimide propionate solution in step d) is 10 mM.

[0016] Furthermore, the reaction time described in step d) is 3-3.5 h.

[0017] Further, the concentration of the METH antibody solution described in step d) is 1 mg / mL.

[0018] Furthermore, in step d), the amount of dithiobissuccinimide propionate solution and METH antibody solution used per milliliter of Au@Raman signal molecule@Ag solution is 1 μL.

[0019] Furthermore, the incubation time described in step d) is 1.5h-2h.

[0020] Furthermore, the soaking time described in step e) is 1 to 2 hours.

[0021] Further, in step f), the mass ratio of PEI to CTS in the mixed solution is 8:1. This invention modifies PEI onto a PVDF membrane through chemical covalent bonding to improve its protein immobilization ability.

[0022] Furthermore, the stirring time described in step f) is 10 hours.

[0023] Further, the concentration of tannic acid in the Tris-HCl buffer solution containing tannic acid described in step g) is 10 mM. In this invention, the prepared PVDF-PEI membrane is immersed in a solution containing tannic acid to improve the membrane's performance stability, enabling it to better and more stably immobilize proteins.

[0024] Furthermore, the soaking time at room temperature described in step g) is 12~24h.

[0025] Further, in step h), the METH antigen is mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution at a volume ratio of 1:1:2, and then the PVDF-PEI composite membrane is added and soaked for 2 hours.

[0026] The antigen-coupled PVDF-PEI membrane-type SERS sensor obtained above can be used for ultra-trace detection of methamphetamine and its derivatives in liquid samples such as domestic sewage, lake water, seawater, saliva and serum.

[0027] This invention develops an antigen-coupled PVDF-PEI membrane-type SERS sensor based on surface-enhanced Raman scattering (SERS) technology for ultra-trace detection of methamphetamine (METH) and its derivatives in samples. It mainly consists of two parts: (1) SERS immunoprobe is synthesized by a layer-by-layer modification method, that is, 4-mercaptobenzoic acid (MBA) is selected as a signal molecule to modify the surface of gold nanoparticles (Au NPs), and then the added silver nitrate (AgNO3) is reduced to silver (Ag) through a redox reaction and coated on the surface of Au@MBA to form a silver shell, thus obtaining core-shell structured nanoparticles Au@MBA@Ag. This process can significantly enhance the signal of the signal molecule MBA between the Au nanospheres and the Ag layer. Then, dithiobissuccinimide propionate (DSP) is used as a crosslinking agent to connect the methamphetamine (METH) detection antibody to the surface of Au@MBA@Ag nanoparticles, thereby obtaining the SERS immunoprobe. (2) The PVDF membrane was modified by solution immersion method. The PVDF membrane was pretreated and then immersed in a mixed solution of polyethyleneimine (PEI) and 3-chloropropyltrimethoxysilane (CTS) to modify the PVDF membrane. Then, it was transferred to tannic acid (TA) solution to stabilize the membrane performance, thus obtaining a PVDF-PEI composite membrane with stable performance and surface modification of PEI. Then, METH-bovine serum albumin (METH-BSA) as METH antigen was mixed with a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to incubate the METH antigen on the PVDF-PEI membrane, thus obtaining an antigen-coupled PVDF-PEI composite membrane.

[0028] During detection, the test solution and the SERS immunoprobe are mixed in equal volumes. If the target substance METH is present in the test solution, the METH in the test solution will compete with METH-BSA incubated on the PVDF-PEI membrane for recognition and binding to the SERS immunoprobe, forming a nuclear satellite-like SERS sensing platform. The SERS immunoprobe contains the Raman signal molecule MBA, and the characteristic peaks of MBA (1074, 1583 cm⁻¹) are obtained. -1 The SERS spectral signal intensity at the membrane surface can be used to quantitatively detect the METH concentration in the sample solution. As the METH concentration in the test solution increases, the SERS signal intensity on the membrane surface decreases, and vice versa.

[0029] The significant advantages of this invention are: The detection linear range of the sensor obtained by this invention is 1 mg / mL - 10 mg / mL. -8 mg / mL, linear correlation coefficient R 2 With a detection limit as low as 10 pg / mL and a value of 0.976, this sensor exhibits excellent anti-interference capabilities, enabling ultra-trace detection of methamphetamine and its derivatives in complex biological environments. Furthermore, the sensor developed in this invention can detect the target substance within 30 minutes, making it suitable for rapid on-site screening of METH in environmental and biological samples. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the fabrication process of the PVDF-PEI membrane-type SERS sensor of the present invention and its application in the detection of methamphetamine (METH).

[0031] Figure 2 The images shown are transmission electron microscope (TEM) images of gold nanoparticles (A) and Au@MBA@Ag (B) prepared in the examples, energy dispersive spectroscopy (EDS) elemental distribution map of Au@MBA@Ag (C), ultraviolet-visible absorption spectra (D), zeta potential map (E), particle size distribution map (F), surface enhanced Raman scattering (SERS) spectrum (G), and standard deviation map of Raman signal of Au@MBA@Ag (H).

[0032] Figure 3 Transmission electron microscopy (TEM) images (A, B) of the PVDF-PEI film-type SERS sensor before and after the addition of the nanoprobe (C, D), and the relative standard deviation of the Raman signal on the PVDF-PEI film (E).

[0033] Figure 4 Methamphetamine (METH) in concentrations of 1 mg / mL to 10⁻ 8 Surface-enhanced Raman scattering (SERS) spectra corresponding to the concentration range of mg / mL (A), linear relationship between METH concentration and SERS spectral intensity (B), incubation time gradient of nanoprobe on SERS sensor (C), and 1583 cm⁻¹ -1 The corresponding signal intensity change (D), and the comparison of SERS spectra of different small molecule illicit drugs detected by the SERS sensor (E) and 1583 cm⁻¹. -1 The corresponding signal strength difference (F).

[0034] Figure 5 Comparison of surface-enhanced Raman scattering (SERS) responses in sewage, lake water, saliva, and serum samples with and without methamphetamine (METH).

[0035] Figure 6This is a performance comparison chart for METH detection before and after PVDF membrane treatment. Detailed Implementation

[0036] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto. Example

[0037] I. Reagents Methamphetamine (METH). METH antibody (METH-Abs) and METH antigen (METH-BSA) were purchased from Shanghai LONGi Biotechnology Co., Ltd. Commercially available PVDF microfiltration membrane (MF, average pore size 0.22 μm). Chloroauric acid (HAuCl4), sodium hydroxide (NaOH), tannic acid (TA), silver nitrate (AgNO3), and trisodium citrate dihydrate (TCD, (Na)3C6H5O7-2H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. Isopropanol was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Merck Biosciences, Germany. Bovine serum albumin (BSA, lyophilized, ≥96%), Tris-HCl (pH=7.8), and Tris-HCl (pH=7.4) were also available. Ascorbic acid (AA) was purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). 3-Chloropropyltrimethoxysilane (CTS), polyethyleneimine (PEI, 50% (w / v) aqueous solution), and 4-mercaptobenzoic acid (4-MBA) were purchased from Shanghai Maclean Biochemical Co., Ltd. (Shanghai, China). Ultrapure (up) water (resistivity 18.2 MΩ cm) used throughout the process was generated by a Milli-Q gradient system.

[0038] II. Sensor Construction 1) Preparation of specific immune probes a) Synthesis of gold nanoparticles (AuNPs): 1 mL of 1 wt% chloroauric acid solution was added to 99 mL of ultrapure water and stirred vigorously while heating. When the solution boiled, 1.5 mL of 1 wt% sodium citrate solution was quickly added. After the solution changed from pale yellow to wine red and the color stabilized, the heating and stirring continued for 15 min. After cooling to room temperature, the AuNPs solution was obtained and stored for later use. b) Synthesis of Au@Raman signal molecule solution: Take 10 mL of AuNPs solution, add 10 μL of 1 mM Raman signal molecule 4-MBA in ethanol solution, incubate for 0.5~1 h, then centrifuge at 8500 rpm for 10 min to remove excess Raman signal molecules. The resulting precipitate is resuspended in ultrapure water to obtain Au@MBA solution.

[0039] c) Synthesis of Au@MBA@Ag solution: Take 10 mL of Au@MBA solution, and add 0.01 mL of 0.1 mM sodium hydroxide solution, 0.1 mL of 1 wt% sodium citrate solution, 1 mL of 10 mM ascorbic acid solution and 1 mL of 1 mM silver nitrate solution in sequence. After stirring for 15 min, centrifuge at 8000 rpm for 10 min to remove the supernatant. Resuspend the precipitate in ultrapure water to obtain Au@MBA@Ag solution, which is stored for later use.

[0040] d) Synthesis of specific nanoprobe solution: Take 2 mL of Au@MBA@Ag solution, add 2 μL of 10 mM dithiobissuccinimide propionate (DSP) solution, react for 3-3.5 h, centrifuge at 7500 rpm for 8 min to remove unreacted DSP, and resuspend the resulting precipitate in 1 mL of water, then add 2 μL of 1 mg / mL METH antibody solution, and incubate at 37 ℃ for 1.5 h-2 h, centrifuge at 7000 rpm for 8 min to remove antibodies not linked to Au@MBA@Ag, then add 500 μL of 0.1% wt bovine serum albumin (BSA) solution for resuspending and blocking for 0.5 h to obtain specific nanoprobe solution.

[0041] 2) Preparation of antigen-conjugated PVDF-PEI composite membrane e) Pretreatment of polyvinylidene fluoride (PVDF) membrane: Cut the original PVDF membrane into circular pieces with a diameter of 0.5 cm, then immerse them in isopropanol for 1-2 hours to clean the membrane surface and disperse the pores. Remove the immersed PVDF membrane and rinse it repeatedly with running ultrapure water several times to remove the isopropanol adhering to the surface. Continue immersion in ultrapure water for at least 30 minutes to wash away any remaining isopropanol adhering to the pores. The pretreated PVDF membrane is then obtained.

[0042] f) Preparation of PVDF-PEI membrane: 1.6 g of polyethyleneimine (PEI) and 0.2 g of 3-chloropropyltrimethoxysilane (CTS) were added to 0.3 mL of ultrapure water to prepare a mixed solution. The mixed solution was placed at room temperature and stirred for 20 min until it turned milky white. The pretreated PVDF membrane was then immersed in the mixed solution and stirred in an open container at room temperature for 10 h. The PVDF-PEI membrane was then rinsed multiple times with ultrapure water to remove excess solutes from the membrane, thus obtaining the PVDF-PEI membrane.

[0043] g) Modification of PVDF-PEI membrane: Add 0.2g of tannic acid (TA) to 0.1mL of Tris-HCl buffer to prepare TA solution. Then place the obtained TA solution in an open container at room temperature and soak the prepared PVDF-PEI membrane in the solution overnight. Rinse several times with ultrapure water to remove excess solution. Then place the membrane in an oven at 60℃ for 0.5h to dry, and obtain a modified PVDF-PEI composite membrane with stable performance.

[0044] h) Antigen coupled to PVDF-PEI membrane A 0.2 M solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and a 0.4 M solution of N-hydroxysuccinimide (NHS) were prepared separately. The three solutions were then mixed at a volume ratio of 1:1:2 for METH antigen (METH-BSA) to EDC and NHS solutions. The resulting PVDF-PEI composite membrane was then immersed in the mixture for 2 hours to covalently fix METH-BSA onto the PVDF-PEI composite membrane via amide bonds, thus obtaining an antigen-coupled PVDF-PEI composite membrane.

[0045] III. SERS Detection 1) SERS detection of METH standard solution Prepare concentrations of 1 mg / mL to 10 mg / mL -8 METH standard solution at mg / mL was prepared. 2 μL of each concentration standard solution was mixed with 2 μL of specific immunoprobe and incubated. The mixture was then dropped onto the antigen-conjugated PVDF-PEI composite membrane and allowed to stand for 15-30 min until the reaction was complete. The membrane was then rinsed three times with ultrapure water to remove any unreacted specific immunoprobes attached to the membrane. The reacted composite membrane was then air-dried at room temperature for 10 min, and SERS was detected using a portable Raman spectrometer to examine the changes in SERS signal intensity on the membrane caused by changes in the concentration of the target substance.

[0046] 2) SERS detection of actual samples Take 5 mL each of domestic sewage, lake water, saliva, and serum samples, group them by number, and then add a solution with a concentration of 10. -3 mg / mL ~10 -5 Prepare a mg / mL METH standard solution to simulate contaminated samples (all simulated samples should be stored at 4°C and used within one week). Process the simulated samples according to the detection method for the METH standard solution and record the SERS signal intensity.

[0047] Depend on Figure 2Mid-transmission electron microscopy (A, B) shows that the prepared Au NPs have a particle size of approximately 20 nm, and the Au@MBA@Ag particles have a particle size of approximately 40 nm. EDS (C) image shows that the Ag shell encapsulates the Au core, with Raman signal molecules embedded within it (confirmed by sulfur). UV spectroscopy (D) shows that the synthesis of the Ag shell introduces a new absorption peak at 383 nm, and the Au peak blue-shifts to 510 nm, while antibody functionalization causes a red-shift. Zeta potential (E) also shows a significant increase in potential with Ag shell deposition and antibody coupling. Particle size and Raman signal characterization (F, G) show that the core-shell particles grow layer by layer, and the Raman signal is enhanced with Ag shell deposition, while antibody functionalization causes a slight decrease in signal. This confirms the successful synthesis and functionalization of the core-shell structure. Furthermore, the Raman signal of the nanoparticles prepared from different batches is stable (H), indicating the successful preparation of a highly sensitive and stable SERS immunoprobe.

[0048] Depend on Figure 3 As can be seen, after modifying the PVDF-PEI composite membrane with METH-BSA, its surface exhibits irregular voids and a smooth skeletal surface (A); while after adding the SERS immunoprobe to the membrane and incubating it, many transparent core-shell nanoparticles appeared on the membrane surface (B). Meanwhile, the SERS spectrum obtained by modifying the PVDF-PEI membrane with only METH-BSA shows a coarse, noisy signal (C), while the SERS immunoprobe specifically recognizes and binds to the incubated METH-BSA, "grabbing" the probe onto the membrane, thus allowing the detection of a smooth and significant SERS spectral signal on the membrane (D). To test the uniformity of the SERS spectral signal intensity on the membrane, arbitrary regions on the membrane incubated with the SERS immunoprobe were selected under a microscope, and the SERS signal intensity at 15-300 points in that region was measured, with a 1583 cm⁻¹ sample taken. -1 The peak value at the location was characterized (E). The results showed that the relative standard deviation (RSD) of the Raman signal at different locations in the same region was only 4.12%, indicating that the PVDF-PEI film has excellent SERS signal uniformity after treatment.

[0049] SERS spectra of METH standard sample solutions of different concentrations participating in immune competition were collected using a portable Raman spectrometer. The results are as follows: Figure 4 As shown in the figure. It can be seen from the graph that as the concentration of METH decreased from 1 mg / mL to 10... -8 At a concentration of mg / mL, the spectral signal intensity of the SERS immunoprobe gradually increased (A). Using the signal molecule MBA at 1583 cm⁻¹... -1 The absolute peak intensity at a given point was used to perform linear analysis on different concentrations of METH. Based on the 3σ principle, the detection limit was 10 fg / mL, where the linear regression value (R0) was [value missing]. 2The result is 0.9760, indicating that this SERS sensor can achieve a range of 1-10 for METH. -8 Dynamic detection over a wide linear range of mg / mL was achieved. To evaluate the detection efficiency of this SERS sensor, an equal volume of SERS immunoprobe was mixed with a certain concentration of METH standard sample solution and then dropped onto a PVDF-PEI membrane incubated with METH-BSA. The incubation time of the SERS immunoprobe was monitored, and changes in the SERS spectral signal intensity on the membrane were detected using a portable Raman spectrometer. The results (C, D) showed that when the mixed solution was dropped onto the membrane and allowed to bind for 30 minutes, the competitive immunization process was maximized, demonstrating that under identical conditions, this timeframe yielded the highest detectable SERS spectral signal intensity. Furthermore, when the immunoprobe was dropped onto the PVDF-PEI membrane, allowed to stand for 1 minute, and then rinsed with UP water to remove unbound probes, a stable and high-intensity SERS signal could still be detected on the membrane. In other words, the novel SERS sensor used in this invention enables "on-demand detection" for on-site clinical testing. Furthermore, when using this SERS sensor to detect METH and other small molecule illicit drugs such as morphine and marijuana, it exhibits excellent specificity for METH (E, F).

[0050] Figure 5 To investigate the Raman spectral differences of the fabricated SERS sensor in simulated biological environments with and without METH, the results showed that the SERS sensor could still detect a strong SERS signal in simulated samples without METH, indicating that the constructed SERS sensor has good signal output capability in complex matrices. Meanwhile, a significant SERS signal attenuation was observed in simulated samples containing METH, demonstrating that the sensor can still achieve high sensitivity and specificity for METH detection under various complex backgrounds.

[0051] To further evaluate the accuracy of the novel SERS sensor in detecting METH content in environmental wastewater samples, a spiked recovery experiment was conducted. The calculated recoveries ranged from 99.69% to 100.9%, with RSD values ​​all less than 0.05%. This demonstrates that the novel SERS sensor possesses excellent detection and quantification capabilities and can be used in on-site clinical applications.

[0052] Finally, to further demonstrate the performance of this SERS sensor, the performance of antigen immobilized on an unmodified PVDF membrane for METH detection was compared. The results are as follows: Figure 6 As shown. By Figure 6It is evident that, under identical conditions, when immobilizing METH-BSA antigen on both the original PVDF membrane and the treated PVDF-PEI membrane, the treated PVDF-PEI membrane exhibits superior SERS signal response. While the original PVDF membrane also shows an SERS signal response, its strong hydrophobicity weakens its antigen immobilization ability, resulting in fewer target sites for antibody binding. Overall, its METH detection performance is weaker, making it prone to misinterpretation of results in the presence of low concentrations of METH.

[0053] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. An antigen-coupled PVDF-PEI membrane-type SERS sensor, characterized in that, The SERS sensor comprises two parts: a specific immune probe and an antigen-conjugated PVDF-PEI composite membrane. Its preparation method includes the following steps: 1) Preparation of specific immune probes a) Synthesis of AuNPs: Chloroauric acid solution was added to water and stirred vigorously and heated. When the solution boiled, sodium citrate solution, a reducing agent, was quickly added. The mixture was heated and stirred for 15 minutes and then cooled to room temperature to obtain AuNPs solution. b) Synthesis of Au@Raman signal molecule solution: Add Raman signal molecule solution to the obtained AuNPs solution and incubate to obtain Au@Raman signal molecule solution; c) Synthesis of Au@Raman signal molecule@Ag solution: Sodium hydroxide solution, sodium citrate solution, ascorbic acid solution and silver nitrate solution were added sequentially to the obtained Au@Raman signal molecule solution. After stirring and reacting thoroughly, the supernatant was removed by centrifugation, and the precipitate was resuspended in water to obtain Au@Raman signal molecule@Ag solution. d) Synthesis of specific nanoprobe solution: After adding dithiobissuccinimide propionate solution to the obtained Au@Raman signal molecule@Ag solution and reacting, METH antibody solution was added, and then incubated at 37°C. After resuspending and blocking with bovine serum albumin, the specific nanoprobe solution was obtained. 2) Preparation of antigen-conjugated PVDF-PEI composite membrane e) Pretreatment of PVDF membrane: After cutting the PVDF membrane to the required size, immerse it in isopropanol, and then rinse it repeatedly with running water to obtain the pretreated PVDF membrane. f) Preparation of PVDF-PEI membrane: The pretreated PVDF membrane was immersed in a mixed solution of polyethyleneimine and 3-chloropropyltrimethoxysilane and stirred at room temperature to obtain PVDF-PEI membrane; g) Modification of PVDF-PEI membrane: The prepared PVDF-PEI membrane was immersed in Tris-HCl buffer containing tannic acid at room temperature and then dried to obtain the modified PVDF-PEI composite membrane. h) Antigen conjugation on PVDF-PEI membrane: METH antigen is covalently immobilized onto PVDF-PEI composite membrane to obtain antigen-conjugated PVDF-PEI composite membrane.

2. The antigen-coupled PVDF-PEI membrane-type SERS sensor according to claim 1, characterized in that: The volume ratio of chloroauric acid solution, water and sodium citrate solution used in step a) is 1:99:1.5, wherein the concentration of chloroauric acid solution is 1wt% and the concentration of sodium citrate solution is 1wt%.

3. The antigen-coupled PVDF-PEI membrane-type SERS sensor according to claim 1, characterized in that: In step b), the amount of Raman signal molecule solution added is calculated as 1 μL per milliliter of AuNPs solution; the concentration of the Raman signal molecule solution is 1 mM, and the Raman signal molecule used is 4-mercaptobenzoic acid; the incubation time is 0.5 to 1 h.

4. The antigen-coupled PVDF-PEI membrane-type SERS sensor according to claim 1, characterized in that: In step c), 10 μL of 0.1 mM sodium hydroxide solution, 100 μL of 1 wt% sodium citrate solution, 1 mL of 10 mM ascorbic acid solution and 1 mL of 1 mM silver nitrate solution are added to every 10 mL of Au@Raman signal molecule solution; the stirring reaction time is 15 min.

5. The antigen-coupled PVDF-PEI membrane-type SERS sensor according to claim 1, characterized in that: Step d) The amount of dithiobissuccinimide propionate solution and METH antibody solution used per milliliter of Au@Raman signal molecule@Ag solution is 1 μL, wherein the concentration of the dithiobissuccinimide propionate solution is 10 mM and the concentration of the METH antibody solution is 1 mg / mL; the reaction time is 3-3.5 h and the incubation time is 1.5 h-2 h.

6. The antigen-coupled PVDF-PEI membrane-type SERS sensor according to claim 1, characterized in that: In step f), the mass ratio of polyethyleneimine to 3-chloropropyltrimethoxysilane in the mixed solution is 8:1, and the stirring time is 10 hours.

7. The antigen-coupled PVDF-PEI membrane-type SERS sensor according to claim 1, characterized in that: In step g), the concentration of tannic acid in the Tris-HCl buffer solution containing tannic acid is 10 mM, and the soaking time at room temperature is 12-24 h.

8. The antigen-coupled PVDF-PEI membrane-type SERS sensor according to claim 1, characterized in that: In step h), the METH antigen is mixed with EDC solution and NHS solution in a volume ratio of 1:1:2, and then the PVDF-PEI composite membrane is added and soaked for 2 hours.

9. The application of an antigen-coupled PVDF-PEI membrane-type SERS sensor as described in claim 1 in ultra-trace detection of methamphetamine and its derivatives in liquid samples.

10. The application according to claim 9, characterized in that: The liquid samples include domestic sewage, lake water, seawater, saliva, and serum samples.

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