Aluminum ion-histidine coordination SERS (Surface Enhanced Raman Scattering) immunosensing method for high-sensitivity detection of alpha fetoprotein

Abstract

The invention discloses an aluminum ion-histidine coordination SERS (Surface Enhanced Raman Scattering) immunosensing method for high-sensitivity detection of alpha fetoprotein. According to the method, a nitrocellulose membrane (NC membrane) is used as a substrate to fix an AFP capture antibody, and gold nanoparticles (Au NPs) co-modified by histidine (His) and 4-mercaptobenzoic acid (4-MBA) are used for marking a second antibody to construct an SERS probe, so that a sandwich immune complex is formed. Au NPs are induced to directionally gather on the surface of the membrane to form a high-density SERS'hot spot 'through the coordination effect of an imidazole ring of Al < 3 + > and His. The hot spot density is dynamically optimized by regulating and controlling the aluminum ion assembly cycle times, and accurate signal amplification is achieved. The detection limit of the method reaches 0.043 pg / mL (5 cycles), the linear range spans 3 orders of magnitude (0.1 pg / mL to 0.2 ng / mL), the recovery rate in a serum sample is 94.9% to 107.5%, and the method has excellent repeatability and anti-interference performance. The problem of uncontrollable construction of solid-phase SERS hot spots is solved, and a high-sensitivity detection tool which is easy and convenient to operate and adjustable in performance is provided for early diagnosis of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to an immunosensing method based on surface-enhanced Raman scattering (SERS) and aluminum ion-induced nanoassembly technology, for the highly sensitive detection of trace biomarkers. Background Technology

[0002] Alpha-fetoprotein (AFP) is an important tumor marker with significant clinical value in the early diagnosis and disease monitoring of various malignant tumors, including hepatocellular carcinoma and ovarian cancer. AFP levels are extremely low in normal adults, but significantly elevated in disease states; therefore, highly sensitive, rapid, and accurate quantitative detection is crucial.

[0003] Currently, routine detection methods for AFP include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and electrochemical sensing technology. Although these methods have good sensitivity and specificity, they generally suffer from problems such as cumbersome operation, long time, and strong dependence on experimental conditions, which limit their application in rapid screening and on-site detection.

[0004] In recent years, surface-enhanced Raman spectroscopy (SERS) has become a powerful tool in the field of tumor marker detection due to its high sensitivity, rapid response, and molecular fingerprint specificity. Combining SERS with immune recognition mechanisms to construct a SERS immunosensing platform can achieve highly specific and sensitive detection of target protein molecules. Its basic principle is to enrich nanoprobes carrying Raman signal molecules onto the sensing platform through specific antigen-antibody binding, and then amplify the signal using "hotspot" structures.

[0005] This invention establishes a SERS immunosensing method based on ion-induced nanoassembly for sensitive detection of AFP. This method utilizes the synergistic effect of antibody recognition and ion assembly amplification to improve detection performance. Compared with traditional SERS sensors or enzyme-catalyzed amplification strategies, the ion-induced assembly platform constructed in this invention has advantages such as simple operation, strong controllability, and no need for enzyme intervention, demonstrating broad application prospects in on-site detection and early diagnosis of tumor markers. Summary of the Invention

[0006] This invention designs a SERS immunosensor based on antigen-antibody recognition. Utilizing the aluminum ion-induced assembly effect of gold nanoparticles, combined with 4-MBA-modified gold nanoparticles to form nano-hot spots, it achieves highly sensitive detection of AFP (alpha-fetoprotein). This method involves immobilizing a primary antibody on a nitrocellulose membrane (NC membrane) to capture the AFP antigen, and then using a secondary antibody labeled with histidine and 4-MBA-modified gold nanoparticles to bind to the antigen, forming a typical sandwich structure. The aluminum ion-induced assembly process further enhances the SERS signal. Specifically:

[0007] (1) Preparation of capture substrate: In this invention, a nitrocellulose membrane (NC membrane) is used as a substrate to fix the AFP primary antibody on the membrane surface by chemical adsorption or physical adsorption, and an immune substrate for capturing AFP antigen is successfully constructed.

[0008] (2) SERS probe construction: This invention uses gold nanoparticles (Au NPs) modified with histidine (His) and 4-mercaptobenzoic acid (4-MBA) as SERS probes. Gold nanoparticles not only possess excellent surface enhancement effects, but also interact with aluminum ions (Al) via histidine... 3+ The coordination effect of the nanoparticles further optimizes the assembly of nanoparticles on the film surface, forming a high-density nano-hotspot structure. SEM characterization revealed that His / 4-MBA@Au NPs have a uniformly distributed surface morphology and can provide a stable SERS signal.

[0009] (3) Immune Response Mechanism: This invention utilizes the immune recognition reaction of antibody antigens for AFP detection. Target antigen capture is achieved through a sandwich immune complex structure, which comprises: (a) a primary antibody immobilized on a nitrocellulose membrane (NC membrane); (b) the captured target antigen (AFP); and (c) a secondary antibody that binds to the target antigen, modified with gold nanoparticles (AuNPs), histidine (His), and the Raman reporter molecule 4-mercaptobenzoic acid (4-MBA). The formation of SERS hotspots is achieved by adding an aluminum ion solution to the sandwich complex system, utilizing the specific coordination between aluminum ions and histidine to induce the aggregation and assembly of histidine-modified gold nanoparticles on the NC membrane surface.

[0010] (4) Ion-induced assembly and hotspot generation: The innovation of this invention lies in the introduction of aluminum ions (Al... 3+This process induces the self-assembly of gold nanoparticles. Under the influence of aluminum ions, the imidazole ring on histidine coordinates with aluminum ions, promoting the aggregation of gold nanoparticles on the membrane surface to form a dense "hot spot" structure, thereby significantly enhancing the SERS signal. This process is an ion-induced assembly effect, which can adjust the assembly density and the number of "hot spots" at different cycle numbers, thereby modulating the SERS signal intensity and improving the detection sensitivity and detection limit.

[0011] (5) SERS Signal Modulation and Performance Characterization: This invention achieves precise modulation of the SERS signal by adjusting the number of ion-induced assembly cycles. Experimental results show that as the number of assembly cycles increases, the aggregation degree and hotspot density of nanoparticles gradually increase, and the SERS signal is significantly enhanced, thereby achieving sensitive detection of lower concentrations of AFP. In addition, the enhancement of the SERS signal also significantly reduces the limit of detection (LOD).

[0012] (6) Reproducibility and Interference Resistance of the Immunosensor: This invention evaluated the reproducibility and interference resistance of the immunosensor. Experimental results show that the sensor has strong anti-interference ability against common interfering substances and can accurately detect AFP in complex samples. Meanwhile, the inter-point SERS signal of the sensor shows almost no change, indicating that the substrate has good reproducibility.

[0013] (7) Analysis of actual samples: The present invention further analyzed AFP in actual samples. The experimental results showed that the sensor has a good recovery rate of AFP in actual samples, which verified its effectiveness and reliability in real environment.

[0014] In summary, this invention provides an innovative SERS immunosensor based on aluminum ion-induced gold nanoparticle assembly, which not only achieves highly sensitive detection of AFP, but also optimizes the sensor's performance through the ion-induced assembly mechanism, exhibiting a low detection limit and good stability, and has broad application prospects.

[0015] The specific technical solution of this invention is as follows:

[0016] An aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein includes the following steps:

[0017] 1) Synthesis of Au NPs: Mix 1 mL of HAuCl4·3H2O solution and 100 mL of ultrapure water and heat to boiling point. Stir continuously for 15 min, then quickly add 1.8 mL of trisodium citrate solution and boil for 15 min. The color turns wine red, indicating the formation of Au NPs. Cool to room temperature for later use.

[0018] 2) Preparation of His / 4-MBA@Au NPs: Add 0.2 mL of His solution and 0.4 mL of 4-MBA solution to 20 mL of Au NPs synthesized in step 1), stir for 2 h to ensure that His and 4-MBA are completely self-assembled on the surface of Au NPs, and obtain histidine and 4-mercaptobenzoic acid modified Au NPs, which are His / 4-MBA@Au NPs;

[0019] 3) The preparation method of SERS nanotags (Ab2-His / 4-MBA@Au NPs) is as follows: Adjust the pH of 5 mL His / 4-MBA@Au NPs to 8-9 with sodium carbonate buffer, then add 50 μg of antibody (Ab2) and stir continuously at room temperature for 2 h; then add bovine serum albumin (BSA) to a final concentration of 0.5% (w / v) to block the free binding sites on Au NPs. After 30 min, centrifuge the mixture at 9000 rpm for 30 min, resuspend the obtained Ab2-His / 4-MBA@Au NPs complex particles in 10 mL of sodium carbonate buffer and store in a refrigerator at 4℃.

[0020] 4) Preparation of capture substrate: The capture antibody (Ab1) was immobilized on a nitrocellulose membrane. Specifically, the nitrocellulose membrane was cut into 0.5cm × 0.5cm pieces, rinsed with ultrapure water, and then equilibrated in PBS buffer. Then, 10 μL of 100 μg / mL capture antibody (Ab1) was dropped onto the nitrocellulose membrane and incubated overnight at 4°C. Afterward, the membrane was washed three times with PBS buffer to remove unbound capture antibody. After drying, the nitrocellulose membrane was gently shaken with 3% BSA for 1 hour to block non-specific binding sites. After washing three times with PBS buffer, the resulting NC membrane was stored at 4°C for later use.

[0021] 5) Immunoassay procedure based on SERS signal amplification: Add 10 μL of alpha-fetoprotein (AFP) sample solution to the NC membrane obtained in step 4), incubate at 37°C for 30 minutes, and wash with PBS buffer to remove any uncaptured residual antigen; subsequently, add 10 μL of SERS nanotags solution to the NC membrane and incubate at 37°C for 1 hour; after washing with PBS buffer, add 10 μL of Al 3+ The solution was incubated with 50 μL His / 4-MBA@Au NPs for 10 min to complete the first amplification. After washing three times with PBS buffer and drying, SERS measurements were performed. For the second amplification, 10 μL of Al was added sequentially. 3+The solution and 50 μL of His / 4-MBA@Au NPs were incubated for 10 min, washed three times with PBS buffer, dried, and then subjected to a second amplification SERS measurement. This cycle was repeated. All SERS measurements were recorded using a Raman spectrometer with a laser intensity of 1.25 mW and an integration time of 10 s.

[0022] Furthermore, in step 1), the concentration of the HAuCl4·3H2O solution is 1 wt%.

[0023] Furthermore, in step 1), the concentration of the trisodium citrate solution is 1 wt%.

[0024] Furthermore, in step 2), the concentration of the His solution is 2.5 mmol / L, and the concentration of the 4-MBA solution is 1 mmol / L.

[0025] Furthermore, in step 3), the concentration of the sodium carbonate buffer solution is 0.05M.

[0026] Furthermore, in step 5), the number of cycles is 1, 3, or 5.

[0027] Furthermore, in step 5), the Al 3+ The concentration of the solution is 2×10 -5 M.

[0028] The beneficial effects of this invention are:

[0029] 1. This invention has high detection sensitivity. It utilizes aluminum ions (Al...) 3+ The specific coordination of gold nanoparticles with histidine (His) induces the directional aggregation of gold nanoparticles at the antigen-antibody binding site, forming a high-density SERS "hot spot," which significantly enhances the Raman signal and enables trace detection of AFP. The detection limit is as low as 8.61 pg / mL after one cycle, 0.095 pg / mL after three cycles, and 0.043 pg / mL after five cycles, meeting the needs of early diagnosis of liver cancer.

[0030] 2. The detection performance of this invention is dynamically adjustable. It innovatively introduces an ion assembly cycle control mechanism. By adjusting the number of aluminum ion interactions, the aggregation density of nanoparticles and the gap between hot spots can be precisely controlled, flexibly optimizing the SERS signal intensity and linear range. The same platform can be adapted to samples of different concentrations (0.1 pg / mL–1000 ng / mL), avoiding batch-to-batch errors caused by the randomness of hot spots in traditional methods.

[0031] 3. This invention possesses strong robustness and practical value. The sensor maintains excellent repeatability and anti-interference performance in complex serum environments, with actual sample detection recovery rates reaching 98.4%-105.3% and 94.9%-107.5%, respectively. The operation process is simple, and it has excellent anti-interference capabilities, providing a reliable tool for point-of-care testing (POCT). Attached Figure Description

[0032] Figure 1 The images show TEM images of Au NPs (A) and His / 4-MBA@Au NPs (B) in Example 1; and FT-IR spectra of 4-MBA powder, His powder, and His / 4-MBA@Au NPs (C).

[0033] Figure 2 A is the UV-Vis spectrum of Au NPs, His / 4-MBA@Au NPs and SERS nanotags in Example 1; Figure 2 B is a graph evaluating the signal stability of SERS nanotags.

[0034] Figure 3 The image shows the EDS elemental image (right) and the mapping image (SEM) (left) of Au elements generated by the aggregation of 1 cycle (A) and 5 cycles (B) in Example 2.

[0035] Figure 4 Is there Al in Example 2? 3+ Characteristic SERS intensity of immune sensors inducing aggregation process.

[0036] Figure 5 In Example 2, three different models were used to detect and analyze different concentrations of AFP. The relationship between SERS intensity and AFP concentration and the standard curve (D) were obtained from the three different models of aluminum ion induced aggregation under 5 cycles (A), 3 cycles (B), and 1 cycle (C).

[0037] Figure 6 In Example 2, 5 cycles (A) and 1 cycle (B) were performed at 1074 cm. -1 The SERS intensity at 1074 cm⁻¹ was used for AFP detection and other interfering antigens (including CEA, PSA, CA 125, CA 19-9, and mixtures); repeatability of the SERS spectra of the sandwich immunoassay structure for 5 cycles of AFP (0.01 ng / mL) (C) and 1 cycle of AFP (10 ng / mL) (D); -1 and 1585cm -1 The relative standard deviation (RSD) of peak intensity at 5 cycles (E) and 1 cycle (F).

[0038] Figure 7 It is Al3+ - Schematic diagram of ultrasensitive detection of alpha-fetoprotein (AFP) by histidine coordination-mediated tunable SERS hotspot. Detailed Implementation

[0039] To better understand the technical solution of the present invention, specific embodiments are provided for further detailed explanation, but the technical solution is not limited thereto.

[0040] Example 1: Preparation method of SERS nanotags (Ab2-His / 4-MBA@Au NPs)

[0041] 1) Synthesis of Au NPs

[0042] Mix 1 mL of 1 wt% HAuCl4·3H2O solution with 100 mL of ultrapure water and heat to boiling point. Stir continuously for 15 min, then quickly add 1.8 mL of 1 wt% trisodium citrate solution and boil for 15 min. The color turns wine red, indicating the formation of AuNPs. Cool to room temperature for later use.

[0043] 2) Preparation of His / 4-MBA@Au NPs

[0044] Add 0.2 mL of His (2.5 mmol / L) and 0.4 mL of 4-MBA (1 mmol / L) dropwise to 20 mL of Au NPs synthesized in step 1), stir for 2 h to ensure that His and 4-MBA are completely self-assembled on the surface of Au NPs, and obtain Au NPs modified with histidine and 4-mercaptobenzoic acid, namely His / 4-MBA@Au NPs.

[0045] The composition and properties characterization of relevant Au NPs and His / 4-MBA@Au NPs are shown in [reference needed]. Figure 1 .

[0046] Figure 1 A is a transmission electron microscope (TEM) image of AuNPs. The TEM image confirms that the prepared AuNPs exhibit a spherical morphology. Figure 1 A). His / 4-MBA@Au nanoparticles were formed by functionalization with histidine (His) and 4-mercaptobenzoic acid (4-MBA). The morphology of the gold nanoparticles remained unchanged, and excellent monodispersity was maintained. Figure 1 B).

[0047] Figure 1 C represents the FT-IR spectra of 4-MBA powder, His powder, and His / 4-MBA@Au NPs. The FT-IR spectra confirm the successful dual functionalization of Au NPs with 4-MBA and histidine. Characteristic benzene ring vibrational bands (1180, 1010, 756, and 538 cm⁻¹) are also shown.-1 The presence of SH stretching peak (2557 cm⁻¹) accompanied by the presence of SH stretching peak. -1 The disappearance of ) was verified by Au-S covalent bonding, confirming the 4-MBA linkage. For histidine coordination, asymmetric / symmetric COO - The dimensions are 1581cm. -1 and 1415cm -1 Carboxylate shoulder (1145cm) -1 ) and NH-related zone (833cm) -1 NH2 swinging; 1499cm -1 NH3 + The deformation together proves that the molecules are fixed.

[0048] 3) Preparation of SERS nanotags (Ab2-His / 4-MBA@Au NPs)

[0049] Adjust the pH of 5 mL of the His / 4-MBA@Au NPs prepared in step 2) to 8-9 using sodium carbonate solution (0.05M), then add 50 μg of antibody (Ab2) and stir continuously at room temperature for 2 h. Next, add BSA to a final concentration of 0.5% (w / v) to block free binding sites on the Au NPs. After 30 min, centrifuge the mixture at 9000 rpm for 30 min, resuspend the Ab2-His / 4-MBA@Au NPs complex particles in 10 mL of sodium carbonate buffer, and store at 4°C for further use.

[0050] The relevant composition and property characterization are shown in Figure 2 .

[0051] Figure 2 The UV-Vis spectra of different complexes formed during the assembly of SERS nanotags were obtained. The prepared Au NP showed a characteristic absorption band at 520 nm (black line). After modification with 4-MBA and His, the absorption band red-shifted slightly to 525 nm (blue line), indicating that functionalization did not induce significant nanoparticle aggregation. After binding with the antibody, a further slight red-shift to 528 nm occurred due to additional surface modification. A characteristic antibody absorption peak appeared at 274 nm, confirming that the alpha-fetoprotein (AFP) antibody was successfully anchored to His / 4-MBA@AuNP (red line). The signal stability of the SERS tags was evaluated, and SERS spectra were collected from 10 randomly selected points. Figure 2 B). The SERS intensity distribution on these spots did not change significantly. 1074 cm -1 The relative standard deviation (RSD) at 1585 cm -1The values ​​were as low as 4.73% and 7.79%, respectively, confirming that the prepared SERS tags have excellent signal stability.

[0052] Example 2: Performance Testing of SERS Sensor

[0053] a. Detection of AFP based on aluminum ion-induced Au NPs aggregation amplification strategy

[0054] 1) The capture antibody (Ab1) was immobilized on a nitrocellulose (NC) membrane. Specifically, the NC membrane was cut into small pieces (0.5cm × 0.5cm), rinsed with ultrapure water, and then equilibrated in PBS buffer. Next, 10 μL of the capture antibody (100 μg / mL) was dropped onto the NC membrane and incubated overnight at 4°C. Afterward, the membrane was washed three times with PBS buffer to remove unbound capture antibody. After drying, the membrane was gently shaken with 3% (m / v) BSA for 1 hour to block non-specific binding sites. After washing three times with PBS buffer, the NC membrane was stored at 4°C for later use.

[0055] 2) Add 10 μL of AFP standard solutions of different concentrations to the NC membrane obtained in step 1), incubate at 37°C for 30 minutes, and wash with PBS buffer to remove any uncaptured residual antigen. Then, add 10 μL of SERS nanotags solution to the NC membrane and incubate at 37°C for 1 hour. After washing with PBS buffer, add 10 μL of 2×10⁻⁶ SERS nanotags solution. -5 M AlCl3 solution and 50 μL His / 4-MBA@Au NPs were incubated for 10 min to complete the first amplification. The cells were washed three times with PBS buffer, dried, and then subjected to SERS measurements. For the second amplification, 10 μL of 2×10⁻⁶ ppm of PBS buffer was added sequentially. -5 After incubating with AlCl3 solution and 50 μL His / 4-MBA@AuNPs solution for 10 min, the samples were washed three times with PBS buffer, dried, and then subjected to a second SERS amplification measurement. This cycle was repeated. All SERS measurements were recorded using a Raman spectrometer with a laser intensity of 1.25 mW and an integration time of 10 s. Furthermore, each sample was measured in triplicate.

[0056] The relevant composition and property characterization are shown in Figure 3-5 .

[0057] Figure 3 SEM imaging revealed a significant morphological transformation: after initial immune complex fixation, a single Al... 3+ A small number of Au NPs (cycle 1, 1) were observed during mediated aggregation cycle amplification. Figure 3 A). After 5 cycles, densely packed NP clusters appear. Figure 3B) confirms the gradual assembly of nanoparticles. EDS mapping further validates this spatial accumulation, where the Au signal intensity and distribution abruptly increase from cycle 1 to cycle 5, indicating that Al 3+ The trigger identified more Au NPs in the sensing area.

[0058] Figure 4 SERS spectroscopy directly correlates aggregation state with signal output: Immune complex recognition (cycle 0) only produces a baseline signal. Figure 4 Cycle 0), while the control sample did not amplify (His / 4-MBA@Au NPs alone or with only Al added). 3+ A negligible SERS response was produced, confirming that amplification was dependent on coordination chemistry. Following amplification, the immunosensor reached a wavelength of 10⁷⁴ cm⁻¹. -1 It exhibits a significantly enhanced characteristic peak (CS stretching) at 1585 cm⁻¹. -1 (benzene ring vibration) Figure 4 ,cycle 1).

[0059] Figure 5 For the corresponding SERS intensity and linear comparison. With increasing AFP concentration, the three cycles at 1074 cm⁻¹... -1 The SERS intensity of the characteristic peak at the location gradually increases ( Figure 5 AC). Using 1074cm -1 SERS intensity versus logarithm of AFP concentration ( Figure 5 A standard curve was plotted to show the relationship between D), with the three cycles exhibiting different response ranges and sensitivities. Specifically, the limit of detection (LOD) for one cycle was 8.61 pg / mL, for three cycles it was as low as 0.095 pg / mL, and for five cycles it was as low as 0.043 pg / mL. Compared with previous studies on AFP detection, this method demonstrates higher sensitivity and lower LOD.

[0060] b. Selectivity and reproducibility detection of SERS sensors

[0061] In the specificity assay, this invention detected other common interfering antigens, including CEA, PSA, CA 125, and CA19-9. The concentration of other analogues was 10 times that of the target antigen, and PBS buffer was used as a negative control; the remaining steps were the same as above.

[0062] For relevant performance testing, please refer to Figure 6 .

[0063] Figure 6 A and B are the specific detection results of AFP in Example 2 of this invention. For example... Figure 6As shown in Figures A and B, only AFP and the mixed standard solution exhibited strong Raman responses during cycles 1 and 5, while the Raman intensities of other interfering antigens did not change significantly. These results demonstrate that the established SERS-based immunosensor exhibits good selectivity for AFP detection.

[0064] Figure 6 CF is a reproducibility study of the sensor response. SERS spectra at 15 random locations on the sensor with AFP concentrations of 0.01 ng / mL (cycle 5) and 10 ng / mL (cycle 1), and SERS spectra at 1074 and 1585 cm⁻¹ at these random locations. -1 Raman intensity at the location. Statistical results are shown in [reference needed]. Figure 6 As shown in E and F, the 1074 and 1585 cm of cycle 5 -1 The signal strength RSD values ​​at the locations were 3.08% and 6.89%, respectively, at 1074 and 1585 cm⁻¹ in cycle 1. -1 The RSD values ​​of the signal strength at the locations were 7.24% and 10.88%, respectively, indicating that the SERS sensor has acceptable reproducibility.

[0065] Example 3: Actual Sample Testing

[0066] Testing of actual samples

[0067] To evaluate the application potential of the developed SERS immunosensor in real samples, AFP standard solutions of different concentrations were added to serum diluted 10-fold. After addition, the samples were tested using the procedure described above.

[0068] The relevant data is shown in Table 1.

[0069] Table 1 shows the analytical results of AFP in actual samples. Serum samples supplemented with 0, 1, 10, and 50 ng / mL AFP were analyzed using cycle 1, and serum samples supplemented with 0, 0.05, 0.10, and 0.20 ng / mL AFP were analyzed using cycle 5. As shown in Table 1, the average recovery rate for cycle 1 was 98.4%–105.3%, and the average recovery rate for cycle 5 was 94.9%–107.5%, with corresponding relative standard deviations (RSDs) of 1.3%–6.0% and 3.3%–7.9%, respectively, indicating good accuracy and precision in the quantitative detection of AFP in serum samples.

[0070] Table 1 shows the recovery rate of AFP in serum samples using two analytical models.

[0071]

Claims

1. A highly sensitive aluminum ion-histidine coordination SERS immunosensing method for the detection of alpha-fetoprotein, characterized in that, Includes the following steps: 1) The capture antibody Ab1 was immobilized on a nitrocellulose membrane to obtain the capture substrate NC membrane; 2) Add 10 μL of alpha-fetoprotein (AFP) sample solution to the NC membrane obtained in step 1), incubate at 37°C for 30 minutes, and wash with PBS buffer to remove any uncaptured residual antigen; then add 10 μL of SERS nanotags solution to the NC membrane and incubate at 37°C for 1 hour; after washing with PBS buffer, add 10 μL of Al 3+ The solution was incubated with 50 μL His / 4-MBA@AuNPs for 10 min to complete the first amplification. After washing three times with PBS buffer and drying, SERS measurements were performed. For the second amplification, 10 μL of Al was added sequentially. 3+ The solution and 50 μL of His / 4-MBA@Au NPs were incubated for 10 min, washed three times with PBS buffer, dried, and then subjected to a second amplification SERS measurement. This cycle was repeated. All SERS measurements were recorded using a Raman spectrometer with a laser intensity of 1.25 mW and an integration time of 10 s.

2. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 1, characterized in that, Step 1) Specifically: Cut the nitrocellulose membrane into small pieces of 0.5cm × 0.5cm, rinse with ultrapure water, and then equilibrate in PBS buffer; then, drop 10μL of 100μg / mL capture antibody Ab1 onto the nitrocellulose membrane and incubate overnight at 4°C; then, wash three times with PBS buffer to remove unbound capture antibody, dry, and gently shake the nitrocellulose membrane with 3% BSA for 1 hour to block non-specific binding sites; After washing three times with PBS buffer, the resulting NC membrane was stored at 4°C for later use.

3. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 1, characterized in that, In step 2), the number of cycles is 1, 3, or 5.

4. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 1, characterized in that, In step 2), the Al 3+ The concentration of the solution is 2×10⁻⁶. -5 M.

5. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 1, characterized in that, In step 2), the SERS nanotags are prepared as follows: the pH of 5 mL His / 4-MBA@Au NPs is adjusted to 8-9 with sodium carbonate buffer, then 50 μg of antibody Ab2 is added and stirred continuously at room temperature for 2 h; then BSA is added to a final concentration of 0.5% (w / v) to block the free binding sites on Au NPs. After 30 min, the mixture is centrifuged at 9000 rpm for 30 min, and the resulting Ab2-His / 4-MBA@Au NPs complex particles are resuspended in 10 mL of sodium carbonate buffer and stored in a 4°C refrigerator.

6. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 5, characterized in that, The concentration of the sodium carbonate buffer solution is 0.05M.

7. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 1 or 5, characterized in that, The preparation method of the His / 4-MBA@Au NPs includes the following steps: 1) Synthesis of Au NPs: Mix 1 mL of HAuCl4·3H2O solution and 100 mL of ultrapure water and heat to boiling point. Stir continuously for 15 min, then quickly add 1.8 mL of trisodium citrate solution and boil for 15 min. The color turns wine red, indicating the formation of Au NPs. Cool to room temperature for later use. 2) Preparation of His / 4-MBA@Au NPs: Add 0.2 mL of His solution and 0.4 mL of 4-MBA solution to 20 mL of Au NPs synthesized in step 1), stir for 2 h to ensure that His and 4-MBA are completely self-assembled on the surface of Au NPs, and obtain histidine and 4-mercaptobenzoic acid modified Au NPs, which are His / 4-MBA@Au NPs.

8. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 7, characterized in that, In step 1), the concentration of the HAuCl4·3H2O solution is 1 wt%.

9. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 7, characterized in that, In step 1), the concentration of the trisodium citrate solution is 1 wt%.

10. The aluminum ion-histidine coordination SERS immunosensing method for highly sensitive detection of alpha-fetoprotein according to claim 7, characterized in that, In step 2), the concentration of the His solution is 2.5 mmol / L and the concentration of the 4-MBA solution is 1 mmol / L.

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