Method for detecting aflatoxin B1 by using hot-spot type SERS immunosensor based on tyramine signal amplification

Abstract

The invention belongs to the technical field of harmful substance detection, and particularly relates to a method for detecting aflatoxin B1 through a hot-spot type SERS immunosensor based on tyramine signal amplification. The preparation method specifically comprises the following steps: preparing an SERS active gold nanolayer on the surface of a glass sheet GS by using an APTES functionalized surface assembly method, immobilizing an AFB1 complete antigen on the 4-MBA modified gold nanolayer through EDC / NHS activation, and closing active sites to form the immunocapture substrate. According to the present invention, the AFB1 in the sample and the antigen specificity fixed on the substrate compete for the antibody, the enzyme-labeled secondary antibody is added to react with the antibody, the tyramine functionalized silver nanoparticles are rapidly deposited on the surface of the substrate through the enzyme catalysis to form the SERS hot spot, and the content of the AFB1 in the sample is determined according to the SERS signal response; the hot-spot type SERS immunosensor based on tyramine signal amplification is high in sensitivity, good in reproducibility and high in specificity, and the provided method serves as a novel analysis tool and shows huge potential in the aspect of trace detection of mycotoxin.

Description

Technical Field

[0001] The invention belongs to the technical field of harmful substance detection, and in particular relates to a method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification. Background Art

[0002] Aflatoxin B1 (AFB1) is recognized as one of the most dangerous mycotoxins, known for its hepatotoxicity, immunotoxicity, mutagenicity, carcinogenicity and teratogenicity. It has been classified as a Class 1 human carcinogen by the World Health Organization's International Agency for Research on Cancer (IARC). AFB1 is commonly found in various foods and agricultural products, especially in cereals, peanuts and animal feed. Due to its strong thermal stability, traditional food processing methods often cannot effectively degrade it, posing a significant risk to human health. Therefore, the detection of AFB1 in food and feed is crucial to ensure food safety and public health.

[0003] Various analytical methods have been developed to detect AFB1, including high-performance liquid chromatography, liquid chromatography-mass spectrometry, and liquid chromatography-tandem mass spectrometry. However, these techniques often require specialized laboratory conditions, extensive sample preparation, and expensive instrumentation, which limits their applicability in field or point-of-care settings. Immunoassays and other immunological techniques, such as enzyme-linked immunosorbent assays (ELISAs), are well suited for rapid and efficient detection of mycotoxins and other small molecule contaminants. ELISA is a well-established biochemical gold standard that offers high-throughput processing and strong antibody-antigen affinity, making it widely used for AFB1 detection. However, it still faces limitations, such as moderate sensitivity, narrow linear range, and susceptibility to interference from colored samples, which restricts its use in complex matrices.

[0004] Surface enhanced Raman spectroscopy (SERS) has emerged as a powerful, ultrasensitive vibrational spectroscopic technique. It has many advantages, including minimal sample consumption, rapid detection, high sensitivity, and strong fingerprint specificity. SERS-based immunoassays, which combine SERS tags with antigen-antibody interactions, offer a promising approach for rapid and sensitive detection of low-level biomarkers such as AFB1. Despite the significant advantages of SERS, the sensitivity needs to be further improved due to the diversity and ultratrace levels of analytes in biological samples. To improve the sensitivity of SERS sensors, active substrates with “hotspot” structures have been designed to amplify the SERS signal. In recent years, several ultrasensitive biosensors for trace analysis have been developed using signal amplification techniques, including enzyme-catalyzed signals, nanomaterial signals, and photoelectric signals. Horseradish peroxidase (HRP)-mediated tyramide signal amplification (TSA) has been demonstrated to be an effective signal amplification strategy. In the presence of hydrogen peroxide (H2O2), HRP catalyzes the formation of tyramide (Tyr) covalent bonding sites, resulting in the rapid deposition of a large amount of tyramide at the enzyme reaction site, thereby geometrically amplifying the original signal.

[0005] Based on this, the present invention uses the TSA strategy and combines immunoassay technology to establish a new SERS-based immunoassay for quantifying AFB1 in real samples. The capture substrate was prepared by loading the AFB1 complete antigen on the surface of the Raman-labeled gold substrate, which competed with AFB1 and AFB1 antibody (AFB1-Ab) for immune reaction. The enzyme-labeled secondary antibody (HRP-IgG) binds to the antibody and promotes the enzyme-catalyzed reaction. In the presence of H2O2, tyramide-functionalized silver nanoparticles (Tyr-AgNPs) act as an efficient SERS signal amplifier to achieve accurate and sensitive detection of AFB1. Summary of the invention

[0006] The present invention designed a method to load AuNPs and 4-MBA probes on a glass slide through APTES and incubation, successfully prepared a substrate with uniform SERS signal, realized immune competition reaction by loading AFB1 complete antigen, and characterized its structure, detection mechanism and SERS performance. Specifically:

[0007] (1) The present invention uses APTES modification to assemble AuNPs on a glass slide, incubates 4-MBA to prepare a 4-MBA / AuNPs / GS substrate, and uses SEM to characterize its morphology. The experimental results show that 4-MBA / AuNPs / GS is successfully synthesized and has a uniform SERS signal, which can be used for further modification.

[0008] (2) The present invention constructs an antigen substrate for capture, and the XPS experimental results show that the capture substrate is successfully prepared.

[0009] (3) The detection of AFB1 by the present invention is based on the competitive reaction between AFB1 and AFB1-BSA. When AFB1 is not present, AFB1-Ab is recognized and fixed to the antigen substrate, and then HRP-IgG is added. In the presence of H2O2, HRP catalyzes the deposition of Tyr-AgNPs, thereby enhancing the SERS signal. When AFB1 is added, AFB1-Ab is inhibited, so that the hot spot cannot be generated. Based on the TSA strategy, the SERS immunoassay has a higher sensitivity and a lower minimum detection limit.

[0010] (4) The present invention investigated the uniformity of the substrate, as well as the stability and anti-interference of the immunosensor. The results showed that the 4-MBA / AuNPs / GS substrate had acceptable uniformity, the SERS intensity of the sensor had almost no change within 20 days, and had good anti-interference ability.

[0011] (5) The present invention has a satisfactory recovery rate for the analysis of AFB1 in actual samples.

[0012] The specific technical solutions of the present invention are as follows:

[0013] The present invention synthesizes AuNPs and AgNPs by sodium citrate reduction method. AuNPs were successfully assembled on a glass slide by piranha treatment and APTES amination treatment, and a SERS substrate (4-MBA / AuNPs / GS) was formed after incubation with 4-MBA. Further, AFB1-BSA was loaded on the substrate surface by EDC / NHS activation to prepare a capture substrate. After a competitive reaction, AFB1-Ab and HRP-IgG were captured on the substrate in turn, and after adding H2O2, HRP catalyzed the deposition of tyramide-functionalized AgNPs on the substrate. The TSA strategy enhances the SERS performance of the substrate and further expands its application in the field of small molecule determination.

[0014] A method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification, characterized in that the method comprises the following steps:

[0015] 1) Sample reaction: Add the test solution and AFB1 antibody to the substrate modified with AFB1-BSA / 4-MBA / AuNPs / GS to perform antigen-antibody recognition reaction, and wash with PBST buffer to remove unbound target and antibody;

[0016] 2) Signal amplification: HRP-labeled goat anti-mouse IgG solution was added for incubation, and tyramide-modified silver nanoparticles Tyr-AgNPs and hydrogen peroxide H2O2 solution were further introduced. SERS hotspot structure was generated through HRP-catalyzed reaction, and surface enhanced Raman spectroscopy (SERS) technology was used to detect Raman signals to achieve quantitative analysis of aflatoxin B1.

[0017] Furthermore, in the above-mentioned method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification, in step 2), the concentration of the HRP-labeled goat anti-mouse IgG solution is 5 μg / mL.

[0018] Furthermore, in the above-mentioned method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification, in step 2), the concentration of the H2O2 solution is 0.05M.

[0019] Furthermore, in the above-mentioned method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification, the preparation method of the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate comprises the following steps:

[0020] 1) Synthesis of AuNPs: Under vigorous stirring, 2 mL of sodium citrate solution was added to 100 mL of boiling HAuCl4·3H2O solution for 20 minutes. The solution slowly turned into wine red and cooled to room temperature to obtain an AuNPs aqueous solution.

[0021] 2) Preparation of 4-MBA / AuNPs / GS: The glass slide GS was ultrasonically treated in ethanol, acetone and deionized water for 30 min, respectively, and then dried in an oven at 90 °C; the dried GS substrate was immersed in a freshly prepared piranha solution at 80 °C for 30 min; subsequently, the GS substrate was thoroughly rinsed with deionized water and dried in an oven at 90 °C; then, the GS substrate was vertically immersed in an anhydrous ethanol solution of APTES at room temperature for 1 h; thereafter, the obtained APTES-functionalized GS substrate was subjected to ethanol and deionized water for 5 min, respectively. Ultrasonic treatment; curing the APTES-functionalized GS in an oven at 120°C for 1.5 hours, immersing the prepared APTES-functionalized GS substrate in an AuNPs aqueous solution at room temperature overnight to form an AuNPs layer on the APTES-functionalized GS substrate, namely AuNPs / GS; then rinsing the prepared AuNPs / GS substrate with deionized water and drying with a N2 gas flow; adding 110 μL of 4-MBA solution to the AuNPs / GS substrate, incubating for 30 minutes, rinsing the 4-MBA / AuNPs / GS and drying;

[0022] 3) Preparation of AFB1-BSA / 4-MBA / AuNPs / GS: 10 μL EDC solution, 10 μL NHS solution and 10 μL AFB1-BSA solution were added to the 4-MBA / AuNPs / GS substrate and incubated overnight at 4°C; then the unreacted AFB1-BSA was removed by washing with PBST to obtain AFB1-BSA / 4-MBA / AuNPs / GS; then, 3% BSA was added to mask the bare sites of AFB1-BSA / 4-MBA / AuNPs / GS at 37°C for 1 hour to finally obtain the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate.

[0023] Furthermore, in the preparation method of the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate, in step 1), the concentration of the sodium citrate solution is 1wt%, and the concentration of the HAuCl4·3H2O solution is 0.01wt%.

[0024] Furthermore, in the method for preparing the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate, in step 2), the volume concentration of the APTES anhydrous ethanol solution is 10%.

[0025] Furthermore, in the preparation method of the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate, in step 2), the concentration of the 4-MBA solution is 10 -3 M.

[0026] Furthermore, in the preparation method of the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate, in step 3), the concentration of the EDC solution is 0.2 M, the concentration of the NHS solution is 0.05 M, and the concentration of the AFB1-BSA solution is 10 μg / mL.

[0027] Furthermore, in the above-mentioned method for detecting aflatoxin B1 using a hotspot-type SERS immunosensor based on tyramide signal amplification, the preparation method of the tyramide-modified silver nanoparticles Tyr-AgNPs includes the following steps: adding 50 μL of tyramide solution to 10 mL of AgNPs colloid and stirring for 2 hours; after tyramide is adsorbed on AgNPs, centrifuging the colloidal solution at 4500 rpm for 10 minutes, and resuspending the precipitate in 5 mL of ethanol to obtain Tyr-AgNPs.

[0028] Furthermore, in the method for preparing the tyramine-modified silver nanoparticles Tyr-AgNPs, the concentration of the tyramine solution is 10 -2 M.

[0029] The beneficial effects of the present invention are:

[0030] 1. The SERS substrate prepared by the present invention exhibits good uniformity.

[0031] 2. The present invention introduces a tyramide signal amplification strategy and prepares a highly sensitive TSA-SERS sensor based on an immune competitive reaction, which has potential application value in the detection of AFB1 in food.

[0032] 3. The TSA strategy uses HRP catalysis to form a stable covalent bond between adjacent protein residues and tyramide-functionalized silver nanoparticles, which promotes the formation of hot spots and amplifies the SERS signal. It has the characteristics of good signal repeatability, high sensitivity, and strong anti-interference, and has broad application prospects in small molecule detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the SEM image of AuNPs / GS in Example 1.

[0034] Figure 2 This is the SERS spectrum of 4-MBA / AuNPs / GS in Example 1.

[0035] Figure 3 The results are from 1000 random detection points to 1585 cm in Example 1. -1 Heat map of the SERS intensity of 4-MBA.

[0036] Figure 4 These are the XPS spectra of 4-MBA / AuNPs / GS (a) and AFB1-BSA / 4-MBA / AuNPs / GS (b) in Example 1.

[0037] Figure 5 is the size distribution histogram of AgNPs and Tyr-AgNPs in Example 2.

[0038] Figure 6 This is the Zeta potential diagram of AgNPs, Tyr and Tyr-AgNPs in Example 2.

[0039] Figure 7 is the SERS spectra obtained at different AFB1 concentrations in Example 3 (A); at 1585 cm -1 The corresponding SERS intensity at (B); (B) the inset is at 1585cm -1 The standard curve of the relationship between the SERS intensity at 400 nm and the AFB1 concentration.

[0040] Figure 8(A) is the interference of ZEN, DON, AFG2, AFB2 and AFG1 in the TSA-SERS sensor to detect the specificity of AFB1 in Example 3; (B) is the detection of different concentrations of 1585 cm by AFB1 at different storage times -1 The SERS intensity at .

[0041] Fig. 9 When the concentration of AFB1 in Example 3 was 0.1 ng / mL, (A) is the SERS spectrum and (B) is the 1076 and 1585 cm-1 spectra of 20 points randomly selected from the same sample. -1 (C) is the SERS spectrum and (D) is the band intensity diagram at 1076 and 1585 cm from 8 different SERS immunosensors -1 Band intensity diagram at .

[0042] Fig.10 (A) is a flow chart for the preparation of antigen substrates; (B) is a schematic diagram of the SERS biosensor based on tyramide-induced amplification for the detection of AFB1. DETAILED DESCRIPTION

[0043] In order to better understand the technical solution of the present invention, specific embodiments are further described in detail, but the technical solution is not limited thereto.

[0044] Example 1 Preparation method of AFB1-BSA / 4-MBA / AuNPs / GS capture substrate

[0045] 1) Synthesis of AuNPs

[0046] Under vigorous stirring, 2 mL of sodium citrate solution (1 wt %) was added to 100 mL of boiling 0.01 wt % HAuCl4·3H2O solution for 20 min, and the solution slowly turned into wine red and cooled to room temperature. The obtained AuNPs aqueous solution was stored at 4 °C for subsequent use.

[0047] 2) Preparation of 4-MBA / AuNPs / GS

[0048] A 0.5 cm × 0.5 cm glass slide (GS) was ultrasonically treated in ethanol, acetone, and deionized water for 30 min, respectively, and then dried in an oven at 90 °C. The dried GS substrate was immersed in a freshly prepared piranha solution (30% H2O2 and 96% H2SO4, 3:7) at 80 °C for 30 min. Subsequently, the GS substrate was thoroughly rinsed with deionized water and dried in an oven at 90 °C. Then, the GS substrate was vertically immersed in a 10% (v / v) solution of 3-aminopropyltriethoxysilane (APTES) in anhydrous ethanol for 1 hour at room temperature. Afterwards, the obtained APTES-functionalized GS substrate was ultrasonically treated with ethanol and deionized water for 5 minutes, respectively, to remove any physically adsorbed APTES molecules. Before depositing AuNPs, the APTES-functionalized GS was cured in an oven at 120 °C for 1.5 hours. The prepared APTES-functionalized GS substrate was immersed in the AuNPs aqueous solution at room temperature overnight to form an AuNPs layer (AuNPs / GS) on the APTES-functionalized GS substrate. The prepared AuNPs / GS substrate was then rinsed with deionized water and dried with a N2 stream. In addition, 110 μL of 4-MBA solution (10 -3 M) was added to the AuNPs / GS substrate and incubated for 30 min, then the 4-MBA / AuNPs / GS was rinsed and dried, and the Raman spectra were recorded.

[0049] The composition and properties of 4-MBA / AuNPs / GS are shown in Figure 1 , Figure 2 and Figure 3 .

[0050] Figure 1 is the scanning electron microscope image of AuNPs / GS. Figure 1 It can be clearly seen that the AuNPs film is dense and uniform without obvious aggregation and large gaps, which lays the foundation for providing uniform Raman signals.

[0051] Figure 2 is the SERS spectrum of 4-MBA / AuNPs / GS, Figure 2 Displayed at 1076cm -1 and 1585cm -1 Two major Raman peaks were observed in the spectra, which are attributed to the in-plane ring breathing mode combined with the ν (CS) and aromatic ring ν (CC) vibration modes, respectively.

[0052] Figure 3 1000 random test points to 1585cm -1 Heat map of the SERS intensity of 4-MBA. Figure 3 Shown at 1585cm -1There is almost no significant difference in the SERS intensity distribution between the points, and the relative standard deviation (RSD) is as low as 10.8%, indicating that the 4-MBA / AuNPs / GS substrate has good uniformity.

[0053] 3) Preparation of AFB1-BSA / 4-MBA / AuNPs / GS

[0054] 10 μL of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) hydrochloride carbodiimide (EDC) solution, 10 μL of 0.05 M N-hydroxysuccinimide (NHS) solution, and 10 μL of 10 μg / mL AFB1-BSA solution were added to the 4-MBA / AuNPs / GS substrate and incubated overnight at 4°C. The unreacted AFB1-BSA was then removed by washing with PBST to obtain AFB1-BSA / 4-MBA / AuNPs / GS. Then, 3% BSA was added to mask the bare sites of AFB1-BSA / 4-MBA / AuNPs / GS at 37°C for 1 hour. Finally, the prepared AFB1-BSA / 4-MBA / AuNPs / GS capture substrate was stored at 4°C for further experiments.

[0055] The composition and properties of the related AFB1-BSA / 4-MBA / AuNPs / GS materials can be found in Figure 4 .

[0056] like Figure 4 It is the XPS spectra of 4-MBA / AuNPs / GS and AFB1-BSA / 4-MBA / AuNPs / GS. Figure 4 The obtained wide scan X-ray photoelectron spectra are shown. In both spectra, the characteristic peaks observed at 83eV, 103eV, 284eV, 399eV and 531eV are attributed to gold (Au 4f), silicon (Si 2p), carbon (C 1s), nitrogen (N 1s) and oxygen (O 1s), respectively. Figure 4 As shown in (b), when AFB1-BSA was immobilized on the surface of 4-MBA / AuNPs / GS, the N1s peak increased due to the high nitrogen content in the protein molecule. XPS surface characterization confirmed that the complete antigen of AFB1 was anchored on the surface of 4-MBA / AuNPs / GS.

[0057] Example 2 Preparation of Tyramine-functionalized AgNPs (Tyr-AgNPs)

[0058] The colloidal solution of AgNPs was prepared by the Lee and Meisel method. 50 μL of tyramine solution (10 -2M) was added to 10 mL of AgNPs colloid and stirred for 2 hours. After tyramine was adsorbed on AgNPs, the colloidal solution was centrifuged at 4500 rpm for 10 minutes to remove excess tyramine. The precipitate was resuspended in 5 mL of ethanol to obtain Tyr-AgNPs.

[0059] Related preparation and characterization see Figure 5 and Figure 6 .

[0060] Figure 5 The DLS distribution of AgNPs and Tyr-AgNPs. As shown in the figure, the average particle hydrodynamic sizes of AgNPs and Tyr-AgNPs are 64.4±3.4 and 72.0±3.2nm, respectively. The results show that tyramine molecules can be effectively adsorbed on AgNPs.

[0061] Figure 6 is the Zeta potential of AgNPs and Tyr-AgNPs, and its value changes from -35.9±1.6mV to -21.5±1.2mV. Zeta potential measurement also indicates that tyramine and silver nanoparticles are successfully conjugated.

[0062] Example 3 Performance Testing of Hotspot SERS Immunosensor

[0063] (I) Hotspot SERS immunosensor with tyramide signal amplification for detection of AFB1

[0064] 20 μL AFB1 standard solution and 20 μL AFB1-Ab were added to the prepared AFB1-BSA / 4-MBA / AuNPs / GS substrate and reacted at 37°C for 2 hours. The unreacted AFB1 and AFB1-Ab were washed three times with PBST buffer to remove the unreacted AFB1 and AFB1-Ab. Subsequently, an equal volume of HRP-labeled goat anti-mouse IgG solution (5 μg / mL) was added and incubated at 37°C for 30 minutes. After washing three times, 200 μL Tyr-AgNPs and 10 μL 0.05M H2O2 solution were added and reacted with the fixed HRP substrate at 37°C for 20 minutes. After washing, SERS detection was performed using a Raman spectrometer.

[0065] (II) Selective detection of AFB1

[0066] In the specificity test, AFB1 with a concentration of 10 ng / mL was selected to compare with other common interfering mycotoxins, including aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), deoxynivalenol (DON) and zearalenone (ZEN). The above analogs (100 ng / mL) are 10 times the concentration of AFB1 (10 ng / mL), PBS buffer is used as a negative control, and the rest of the steps are the same as above.

[0067] Related performance test results can be found in Figure 7 , 8 、9.

[0068] Figure 7 A is the corresponding SERS spectrum. As the concentration of AFB1 increases, the -1 The SERS intensity of the characteristic peak at gradually weakens ( Figure 7 B) This phenomenon follows the principle of competitive immunoassay, that is, the higher the AFB1 concentration, the less AFB1-Ab and IgG-HRP bind to the immune substrate, resulting in the failure to form hot spots and low Raman intensity. -1 The SERS intensity and the logarithm of AFB1 concentration ( Figure 7 The standard curve was drawn based on the relationship between the two groups (inset B), which represents a relatively wide response range (0.001-100 ng / mL). The lower LOD (S / N=3) of AFB1 was calculated to be 0.11 pg / mL. The linear equation is y=-3563.64x+12695.01 (R 2 =0.9974, x = Lg [aflatoxin B1 (ng / mL)]). Compared with previous studies on AFB1 detection, this method has higher sensitivity and lower LOD.

[0069] Figure 8 is the specific detection result of AFB1. Figure 8 As shown in A, only the Raman intensity of AFB1 and the mixed standard solution decreased significantly, while the Raman intensity of other interfering mycotoxins did not change significantly. These results indicate that the established SERS-based immunosensor has good selectivity for the detection of AFB1. The stability of the TSA-SERS platform was further tested on different days. Three different concentrations of AFB1, 0.1, 1, and 10 ng / mL, were selected for SERS measurement. Figure 8 As shown in B, at 1585cm -1 The SERS intensity at 240 nm did not change significantly from 0 to 20 days. The comparison results clearly show that the platform has high specificity and outstanding stability.

[0070] Fig. 9This is a study on the reproducibility of the SERS immunosensor response. The SERS spectra of 20 random positions of the sensor when the AFB1 concentration is 0.1 ng / mL and the SERS spectra of 20 random positions at 1076 cm -1 and 1585cm -1 The Raman intensity at , and 8 immunosensors were prepared under the same conditions, using the same concentration of AFB1 for sensing analysis, and the reproducibility between batches was estimated. The statistical results are shown in Fig. 9 B and 9D 1076cm -1 and 1585cm -1 The signal intensities of the sensors were 6.53% and 5.36%, respectively, and the RSD values ​​among different sensors were 7.45% and 5.92%, respectively, indicating that the SERS sensor had acceptable reproducibility.

[0071] Example 4 Actual sample detection

[0072] In the actual sample test, peanuts (2g) and corn (2g) were mixed with 8mL of organic solvent (CH3OH / H2O=7:3 (v / v)). The extract was ultrasonically treated for 30min, centrifuged at 8000rpm for 20min, and the supernatant was collected. Then a certain amount of AFB1 was added to the supernatant. Finally, the Raman spectrum of the sample was measured and the sample recovery rate was calculated.

[0073] See Table 1 for relevant data.

[0074] Table 1 shows the analysis results of AFB1 in actual samples. This method was used to analyze peanut and corn samples with 0, 0.1, 1 and 10 ng / mL AFB1 added. As shown in Table 1, the average recoveries were 95.9%-109.7% and 95.7%-106.1%, and the relative standard deviations (RSDs) were 4.1%-5.5% and 1.9%-7.5%, respectively, indicating good accuracy and precision of quantitative detection in peanut and corn samples.

[0075] Table 1 Detection and analysis results of AFB1 in actual samples

[0076]

Claims

1. A method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification, characterized in that: The method comprises the following steps: 1) Sample reaction: Add the test solution and AFB1 antibody to the substrate modified with AFB1-BSA / 4-MBA / AuNPs / GS to perform antigen-antibody recognition reaction, and wash with PBST buffer to remove unbound target and antibody; 2) Signal amplification: HRP-labeled goat anti-mouse IgG solution was added for incubation, and tyramide-modified silver nanoparticles Tyr-AgNPs and hydrogen peroxide H2O2 solution were further introduced. SERS hotspot structure was generated through HRP-catalyzed reaction, and surface enhanced Raman spectroscopy (SERS) technology was used to detect Raman signals to achieve quantitative analysis of aflatoxin B1.

2. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 1, characterized in that: In step 2), the concentration of the HRP-labeled goat anti-mouse IgG solution is 5 μg / mL.

3. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 1, characterized in that: In step 2), the concentration of the H2O2 solution is 0.05M.

4. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 1, characterized in that: The preparation method of the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate comprises the following steps: 1) Synthesis of AuNPs: Under vigorous stirring, 2 mL of sodium citrate solution was added to 100 mL of boiling HAuCl4·3H2O solution for 20 minutes. The solution slowly turned into wine red and cooled to room temperature to obtain an AuNPs aqueous solution. 2) Preparation of 4-MBA / AuNPs / GS: The glass slide GS was ultrasonically treated in ethanol, acetone and deionized water for 30 min, respectively, and then dried in an oven at 90 °C; the dried GS substrate was immersed in a freshly prepared piranha solution at 80 °C for 30 min; subsequently, the GS substrate was thoroughly rinsed with deionized water and dried in an oven at 90 °C; then, the GS substrate was vertically immersed in an anhydrous ethanol solution of APTES at room temperature for 1 h; thereafter, the obtained APTES-functionalized GS substrate was subjected to ethanol and deionized water for 5 min, respectively. Ultrasonic treatment; curing the APTES-functionalized GS in an oven at 120°C for 1.5 hours, immersing the prepared APTES-functionalized GS substrate in an AuNPs aqueous solution at room temperature overnight to form an AuNPs layer on the APTES-functionalized GS substrate, namely AuNPs / GS; then rinsing the prepared AuNPs / GS substrate with deionized water and drying with a N2 gas flow; adding 110 μL of 4-MBA solution to the AuNPs / GS substrate, incubating for 30 minutes, rinsing the 4-MBA / AuNPs / GS and drying; 3) Preparation of AFB1-BSA / 4-MBA / AuNPs / GS: 10 μL EDC solution, 10 μL NHS solution and 10 μL AFB1-BSA solution were added to the 4-MBA / AuNPs / GS substrate and incubated overnight at 4°C; then the unreacted AFB1-BSA was removed by washing with PBST to obtain AFB1-BSA / 4-MBA / AuNPs / GS; then, 3% BSA was added to mask the bare sites of AFB1-BSA / 4-MBA / AuNPs / GS at 37°C for 1 hour to finally obtain the AFB1-BSA / 4-MBA / AuNPs / GS modified substrate.

5. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 4, characterized in that: In step 1), the concentration of the sodium citrate solution is 1 wt %, and the concentration of the HAuCl4·3H2O solution is 0.01 wt %.

6. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 4, characterized in that: In step 2), the volume concentration of the APTES anhydrous ethanol solution is 10%.

7. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 4, characterized in that: In step 2), the concentration of the 4-MBA solution is 10 -3 M.

8. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 4, characterized in that: In step 3), the concentration of the EDC solution is 0.2 M, the concentration of the NHS solution is 0.05 M, and the concentration of the AFB1-BSA solution is 10 μg / mL.

9. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 1, characterized in that: The preparation method of the tyramine-modified silver nanoparticles Tyr-AgNPs comprises the following steps: adding 50 μL of tyramine solution to 10 mL of AgNPs colloid and stirring for 2 hours; after tyramine is adsorbed on AgNPs, centrifuging the colloid solution at 4500 rpm for 10 minutes, and resuspending the precipitate in 5 mL of ethanol to obtain Tyr-AgNPs.

10. The method for detecting aflatoxin B1 using a hotspot SERS immunosensor based on tyramide signal amplification according to claim 9, characterized in that: The concentration of the tyramine solution is 10 -2 M.

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