Preparation method of SERS / electrochemical two-channel aptamer biosensor for detecting AFB1

Abstract

The invention discloses a preparation method of an SERS / electrochemical two-channel aptamer biosensor for detecting AFB1, and relates to the technical field of biosensors and detection. Comprising the following steps: S1, preparing a capture probe Fe3O4 (at) Au (at) Ag (at) PAMAM (at) Aptamer by adopting a chemical stepwise synthesis method; s2, preparing a signal probe AuNs (at) 4-MBA (at) Ag (at) ssDNA by adopting a chemical stepwise synthesis method; s3, PAMAM is deposited on the surface of the screen-printed electrode through an electro-deposition method, the aptamer is modified through a glutaraldehyde method, and Apt / PAMAM / SPGE is obtained; the screen printing electrode is used as a sensor substrate, and compared with a traditional three-electrode sensor, the sensor is easy to process, low in cost, small in size and easy to integrate. According to the signal probe, gold nanoparticles (AuNPs) grow into gold nanostars (AuNs) in an anisotropic mode, a Raman signal factor wrapped by an AuNs-Ag core-shell structure is prepared, and the excitation intensity of a Raman signal is remarkably improved by means of the multi-metal coupling effect between a capture probe and the signal probe.

Description

Technical Field

[0001] The present invention relates to the field of biosensors and detection technologies, and in particular to a method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1. Background Art

[0002] Aflatoxins (AF) are a class of structurally similar secondary metabolites produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. They are extremely toxic and carcinogenic, among which aflatoxin B1 (AFB1) is particularly carcinogenic.

[0003] Conventional detection methods for AFB1 mainly include chromatography and immunology. Although they have been widely used, chromatography has low sensitivity and is suitable for qualitative analysis but not for quantitative analysis. Although immunology detection methods are easy to operate, false positive results may occur in the same batch of tests, and the testing cost is often high. With the continuous advancement of laser technology and nanomaterial preparation technology, surface plasmon resonance (SPR), surface enhanced Raman (SERS), fluorescence, colorimetry, electrochemistry and other methods are widely used in the field of AFB1 detection, but these detection methods are only single signal outputs, and the interference of environmental conditions often has a greater interference on the signal.

[0004] On the contrary, dual-channel can combine the advantages of two detection methods, use dual-channel signal self-calibration, and have more reliable detection results. As an ideal substitute for antibodies, aptamers have the advantages of low cost, high stability and strong specificity, and are widely used in food safety detection. Gold nanoparticles (AuNPs) are favored because of their translocation sites for binding to a variety of biomacromolecules, significant surface plasmon resonance properties, and excellent optical and photothermal conversion properties. This method is based on the composite system of AuNPs and aptamers. With the help of Raman signal factor 4-MBA, a competitive SERS and electrochemical dual-channel AFB1 aptamer sensor is constructed to achieve rapid and sensitive detection of AFB1 in food. Summary of the invention

[0005] In view of the above problems in the prior art, the present invention provides a SERS / electrochemical dual-channel aptamer sensor for detecting AFB1 and a preparation method thereof. The aptamer sensor is constructed based on the competitive binding between the target and the signal probe, the change in the substrate SERS signal caused by the dissociation of the signal probe, and the change in the electrode DPV peak value, so as to provide a competitive aptamer sensor with high sensitivity, strong specificity and good stability.

[0006] The present invention is achieved through the following technical solution: A method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1, specifically comprising the following steps: S1: Preparation of capture probe Fe3O4@Au@Ag@PAMAM@Aptamer by chemical stepwise synthesis; S2: Preparation of the signal probe AuNs@4-MBA@Ag@ssDNA by chemical stepwise synthesis; S3: PAMAM was deposited on the surface of the screen-printed electrode by electroplating, and the aptamer was modified by glutaraldehyde to obtain Apt / PAMAM / SPGE; the preparation process of Apt / PAMAM / SPGE was as follows: first, the electrode was activated by CV, H2SO4 was added on the surface of the gold electrode, and the mixture was rinsed with deionized water and dried naturally; PAMAM was fixed to the gold electrode by CV, PAMAM was added on the surface of the gold electrode, and the mixture was rinsed with deionized water and dried naturally, and then 10 μL of aptamer standard solution with a concentration of 10 mM was added to the electrode working area and incubated at 37°C; to prevent nonspecific reactions, the mixture was incubated with MCH solution to block the active sites; the mixture was rinsed with 10 mM PBS buffer and ultrapure water in sequence to remove excess MCH, and the mixture was dried naturally to complete the preparation of Apt / PAMAM / SPGE.

[0007] Furthermore, the CV activated the electrode, and 50 μL of H2SO4 with a concentration of 0.5 mol / L was added to the surface of the gold electrode. The CV conditions were: scanning voltage range -1.2-1.2 V, scanning rate 100 mV / s, and scanning step length 2 m; the CV fixed PAMAM to the gold electrode, and 50 μL of PAMAM with a concentration of 0.05 mM was added to the surface of the gold electrode. The CV conditions were: scanning voltage range -0.2-1.1 V, scanning rate 25 mV / s, and scanning step length 2 mV; the MCH solution concentration was 0.5% 50 μL; and the incubation time was 1-3 h.

[0008] Furthermore, the preparation process of Fe3O4@Au@Ag in step S1 is as follows: 62.5 mg Fe3O4 nanoparticles and 2 mg APTES are dissolved in 15 mL ethanol, ultrasonicated for 60 min, and stirred at room temperature for 4 h; amino-functionalized Fe3O4NPs are collected with a magnet, then rinsed with deionized water and ethanol, and dried at 70°C for 4 h; 20 mg amino-functionalized Fe3O4NPs are dissolved in 10 mL ethanol, 20 mL AuNPs are added, and ultrasonicated at 4°C for 60 min. The formed Fe3O4@AuNPs are collected with a magnet, rinsed with deionized water, and 300 μL 2 mM HAuCl4, 3 mL 1% sodium citrate, 600 μL 3 mM hydroquinone, and 300 mg PVP are added in sequence, and stirred at room temperature to complete the gold seed growth, and the formed Fe3O4@Au SP was collected by magnet, rinsed with deionized water, dissolved in 0.2% sodium citrate, and stored at 4°C; 5mL 1% sodium citrate and 200mg PVP were added to the prepared Fe3O4@Au SP in sequence, 300-500μL 30mM AgNO3 was dripped dropwise at a rate of 45s / drop at room temperature, and stirred at room temperature for 4h. The formed Fe3O4@Au@Ag was collected by magnet, rinsed with deionized water, dissolved in 0.2% sodium citrate, and stored at 4°C. The preparation process of the AuNPs nano-gold particles is as follows: 50mL 2mM HAuCl4 was heated to boiling, and then 15mL 1% sodium citrate was added, stirred until the solution turned wine red, the heat source was removed and stirred for 15min, and the nano-gold particle AuNPs suspension was obtained, which was rinsed with deionized water and stored at 4°C.

[0009] Furthermore, the preparation process of Fe3O4@Au@Ag@PAMAM@Aptamer in step S1 is as follows: 1 mL of 2.5% glutaraldehyde is added dropwise to 20 mL of Fe3O4@Au@Ag mixed solution, stirred at room temperature for 2 h, 200 μL of 1 mM PAMAM is added, stirred at room temperature for 2 h, the formed Fe3O4@Au@Ag@PAMAM is collected with a magnet, rinsed with deionized water, 1 mL of 2.5% glutaraldehyde is added to the mixed solution, stirred at room temperature for 1 h, and then 12 μL of 20 mM Aptamer is added, stirred at 37°C for 1 h, and the Fe3O4@Au@Ag@PAMAM@Aptamer capture probe is collected with a magnet. To prevent nonspecific reactions, the active sites are blocked with 0.5% MCH solution.

[0010] Furthermore, it is characterized in that the preparation process of AuNs nanoparticles in step S2 is: add 5 μL 50mM AgNO3 and 5mL 5mM HAuCl4 to 5mL 20mM CTAB, stir, add 570 μL of 1% ascorbic acid by mass, continue stirring until the solution becomes transparent, add 60 μL of prepared AuNPs, stop after the solution turns blue-brown, centrifuge at 4500 rpm / min for 10 min, rinse with deionized water to obtain AuNs, redisperse in 0.2% sodium citrate, and store at 4°C.

[0011] Furthermore, the preparation process of AuNs@4-MBA@Ag in step S2 is as follows: add 200 μL of 5 mM 4-MBA ethanol solution to the prepared AuNs, stir continuously at room temperature for 12 h, centrifuge at 6000 rpm / min for 10 min, and rinse with deionized water to obtain AuNs@4-MBA NPs, add 3 mL of 1% sodium citrate and 90 mg of PVP in sequence, and drip 200-300 μL of 30 mM AgNO3 dropwise at a speed of 15 s / drop, stir at room temperature for 2 h, centrifuge at 4500 rpm / min for 10 min, rinse with deionized water, and the obtained AuNs@4-MBA@Ag is redispersed in 0.2% sodium citrate and stored at 4°C.

[0012] Furthermore, the preparation process of AuNs@4-MBA@Ag@ssDNA in step S2 is: drop 25 μL of 20 mM ssDNA into 5 mL of AuNs@4-MBA@Ag complex, stir at 4°C for 12 h, add 40 μL of 1 mg / mL BSA, and stir at 4°C for 6-8 h; centrifuge at 8000 rpm / min for 10 min, and rinse with deionized water, and store the prepared AuNs@4-MBA@Ag@ssDNA in PBS with a concentration of 0.1 mol / L pH=7 at a temperature of -20°C.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention uses screen-printed electrodes as the sensor substrate. Compared with the traditional three-electrode sensor, it is simple to process, low in cost, small in size and easy to integrate; 2. The present invention utilizes the dual-active site aptamer AFB1 and its complementary chain ssDNA to solve the problem of insufficient binding ability of the single-active site aptamer, so that the sensor has good specificity in the presence of interfering toxins such as AFG1, ZEN, and OTA; 3. The present invention uses PAMAM as a capture probe and electrode surface modification agent, and uses its abundant amino groups on the surface to fix more aptamers, which helps to improve the sensitivity of the sensor. 4. The signal probe of the present invention anisotropically grows gold nanoparticles (AuNPs) into nano-gold stars (AuNs), prepares an AuNs-Ag core-shell structure to encapsulate the Raman signal factor, and utilizes the multi-metal coupling effect between the capture probe and the signal probe to significantly improve the excitation intensity of the Raman signal 5. The sensor constructed by the present invention has excellent detection ability, and the linear range is 10 -5 -10 3 ng / mL, the minimum detection limit is 2×10 -6 ng / mL, and the recovery rate of spiked samples in rice and soybean was good, so it can be used for actual sample detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will now be described by way of example with reference to the accompanying drawings, in which: Figure 1 This is a process flow chart of the biosensor preparation process of Examples 1-3 of the present invention; Figure 2 This is a schematic diagram of the detection principle of the biosensor in Example 4 of the present invention; Figure 3 The SERS response of AFB1 at different concentrations was measured for the present invention; Figure 4 The SERS standard fitting curve of the present invention for measuring different concentrations of AFB1; Figure 5 The DPV response of different concentrations of AFB1 was measured for the present invention; Figure 6 The DPV standard fitting curve of the present invention for measuring different concentrations of AFB1; Figure 7 For the anti-interference test of the present invention; Figure 8 This is a stability test of the present invention. DETAILED DESCRIPTION

[0015] A SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 comprises a screen-printed gold electrode substrate, a capture probe Fe3O4@Au@Ag@PAMAM@Aptamer and a signal probe AuNs@4-MBA@Ag@ssDNA. The screen-printed gold electrode can adsorb more aptamer biomolecules by modifying PAMAM, and the capture probe and the signal probe are sensor signal generating substances. The screen-printed gold electrode substrate, the capture probe Fe3O4@Au@Ag@PAMAM@Aptamer and the AuNs@4-MBA@Ag@ssDNA signal probe of the biosensor constitute a competitive aptamer biosensor. In the absence of a target, the capture probe combines with the signal probe through base complementary pairing to form a complex. With the introduction of AFB1, due to competitive binding, part of the signal probe dissociates, and under the action of a neodymium iron boron magnet, the separated substrate is subjected to SERS detection. When the supernatant is dropped onto the gold electrode that has been linked to the AFB1 aptamer, the dissociated signal probe will re-bind with the aptamer on the electrode, causing the electron transfer rate on the gold electrode surface to change. The change process is electrochemically characterized by differential pulse voltammetry (DPV). The change in SERS / DPV signal intensity is closely related to the AFB1 concentration, ultimately achieving specific quantitative detection of AFB1.

[0016] This embodiment provides a dual-channel sensor for detecting AFB1. The preparation process is as follows: Figure 1 As shown, the principle is as Figure 2 As shown, the preparation method specifically comprises the following steps: Preparation of Fe3O4@Au@Ag@PAMAM@Aptamer: First, AuNPs were prepared by citric acid reduction method. 50mL HAuCl4 (2mM) was heated to boiling, and then 15mL 1% sodium citrate was quickly added and stirred vigorously until the solution turned wine red. The heat source was removed and stirring was continued for 15min. The suspension of gold nanoparticles (AuNPs) was obtained, rinsed with deionized water three times, and stored at 4°C for subsequent use.

[0017] Then, Fe3O4@Au was synthesized by seed-mediated growth method, 62.5mg Fe3O4 nanoparticles and 2mg APTES were dissolved in 15mL ethanol, ultrasonicated for 60min, and vigorously stirred at room temperature for 4h. The amino-functionalized Fe3O4NPs were collected by magnet, then rinsed with deionized water and ethanol three times, and dried at 70℃ for 4h. 20 mg of amino-functionalized Fe3O4 NPs were dissolved in 10 mL of ethanol, and 20 mL of AuNPs were added. The mixture was ultrasonicated at 4 °C for 60 min. The formed Fe3O4@Au NPs were collected with a magnet and rinsed with deionized water. Then, 300 μL of HAuCl4 (2 mM), 3 mL of 1% sodium citrate, 600 μL of hydroquinone (3 mM), and 300 mg of PVP were added in sequence. The mixture was stirred at room temperature for 1 hour, 2 hours, or 3 hours (preferably 2 hours) to complete the gold seed growth. The formed Fe3O4@AuSP was collected with a magnet, rinsed with deionized water, dissolved in 0.2% sodium citrate, and stored at 4 °C. To the prepared Fe3O4@Au SP, 5 mL of sodium citrate (1%) and 200 mg of PVP were added in sequence, and 300 μL, 400 μL or 500 μL (preferably 500 μL) of AgNO3 (30 mM) was added dropwise at a rate of 45 s / drop at room temperature. The mixture was stirred at room temperature for 4 h. The formed Fe3O4@Au@Ag was collected with a magnet, rinsed with deionized water, dissolved in 0.2% sodium citrate, and stored at 4 °C.

[0018] Finally, 1 mL of glutaraldehyde (2.5%) was added dropwise to 20 mL of the Fe3O4@Au@Ag mixture and stirred at room temperature for 1 h. 100 μL of PAMAM (1 mM) was added and stirred at room temperature for 1 h. The formed Fe3O4@Au@Ag@PAMAM was collected by a magnet and rinsed with deionized water. 1 mL of glutaraldehyde (2.5%) was added to the mixture and stirred at room temperature for 1 h. Then 12 μL of aptamer (20 mM) was added and gently stirred at 37 °C for 1 h. The Fe3O4@Au@Ag@PAMAM@Aptamer capture probe was collected by a magnet. To prevent nonspecific reactions, the active site was blocked with 0.5% MCH solution.

[0019] Preparation of AuNs@4-MBA@Ag@ssDNA:

[0020] First, gold nanoparticles (AuNPs) were grown into nano-gold stars (AuNs). 5 μL AgNO3 (50 mM) and 5 mL HAuCl4 (5 mM) were added to 5 mL CTAB (20 mM), stirred for 20 min, and then 570 μL ascorbic acid (1%) was added. Stirring continued until the solution became transparent, and then 60 μL of the prepared AuNPs was added. The mixture was stopped when the solution turned blue-brown. Centrifuged at 4500 rpm / min for 10 min, rinsed with deionized water, and AuNs were obtained. The mixture was redispersed in 0.2% sodium citrate solution and stored at 4°C.

[0021] Secondly, 200 μL of 4-MBA (5 mM) ethanol solution was added to the prepared AuNs, and the mixture was stirred continuously at room temperature for 12 h, centrifuged at 6000 rpm / min for 10 min, and rinsed with deionized water to obtain AuNs@4-MBA NPs. 3 mL of sodium citrate (1%) and 90 mg of PVP were added in sequence, and 200 μL or 250 μL or 300 μL (preferably 250 μL) of 30 mM AgNO3 was added dropwise at a rate of 15 s / drop. The mixture was stirred at room temperature for 2 h, centrifuged at 4500 rpm / min for 10 min, and rinsed with deionized water. The obtained AuNs@4-MBA@Ag was redispersed in 0.2% sodium citrate solution and stored at 4 °C.

[0022] Finally, 25 μL ssDNA (20 mM) was added to 5 mL of AuNs@4-MBA@Ag complex, and gently stirred for 12 h at 4 °C. 40 μL BSA (1 mg / mL) was added and gently stirred for 6 h, 7 h, or 8 h at 4 °C (preferably 7 h). Centrifuge at 8000 rpm / min for 10 min, rinse with deionized water, and store the prepared AuNs@4-MBA@Ag@ssDNA in PBS (pH=7) solution with a concentration of 0.1 mol / L at -20 °C.

[0023] Preparation of Apt / PAMAM / SPGE:

[0024] First, add 50μL H2SO4 (0.5mol / L) on the surface of the gold electrode and activate the electrode by CV. CV conditions: scanning voltage range -1.2-1.2V, scanning speed 100mV / s, scanning step length 2m. Rinse with deionized water and dry naturally for use. Then add 50μL PAMAM (0.05mM) on the surface of the gold electrode and fix PAMAM on the gold electrode by CV. CV conditions: scanning voltage range -0.2-1.1V, scanning speed 25mV / s, scanning step length 2mV. Rinse with deionized water and dry naturally.

[0025] Finally, 10 μL of aptamer standard solution (10 mM) was added to the electrode working area and incubated at 37°C for 1 hour, 2 hours or 3 hours (preferably 2 hours). To prevent nonspecific reactions, 50 μL of 0.5% MCH solution was incubated for 1 hour, 2 hours or 3 hours (preferably 2 hours) to block the active site. The solution was rinsed with 10 mmol / L PBS buffer and ultrapure water in turn, and the excess MCH was removed multiple times. The solution was dried naturally, and Apt / PAMAM / SPGE was successfully prepared.

[0026] Detection of Aflatoxin (AFB1) Take 2 mL of Fe3O4@Au@Ag@PAMAM@Aptamer capture probe and 2 mL of AuNs@4-MBA@Ag@ssDNA Raman signal probe and mix them thoroughly. Stir gently at 37 °C for 2 h. Collect the mixture with a magnet, rinse with deionized water, and add 100 μL of AFB1 standard solution (10 -5 -10 3 ng / mL), incubated at 25°C for 30 min. The sediment separated by the magnet was transferred to a glass slide and characterized by a laser Raman spectrometer. An appropriate amount of supernatant was applied to the treated electrode, incubated for 2 h, rinsed with deionized water, and dried naturally. DPV was detected using an electrochemical workstation in a 10 mM PBS electrolyte containing 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, and 5 mM potassium chloride.

[0027] Data detection of the aptamer sensor prepared in this example: 1. Sensitivity and detection limit Under the optimal parameters, the concentration of 10 -5 -10 3 ng / mL 10 concentration gradients of AFB1 standard solution. As the concentration of AFB1 increases, the magnetic separation substrate -1 The SERS peak at cm gradually weakened, while the electrochemical DPV test current peak of the supernatant showed a trend of gradual strengthening. Figure 3-Figure 6 As shown, we selected 4-MBA at 1570 -1 A linear relationship was established between the SERS peak at cm and the logarithm of the AFB1 concentration. After linear fitting, the regression equation of SERS was Y=-1088X+4721.39, and the detection limit (LOD) was 2×10 -6 ng / mL. Similarly, a linear relationship was established using the peak current of the electrochemical DPV curve and the logarithm of the AFB1 concentration. After linear fitting, the regression equation was Y=86.1X+547.13, and the LOD was 7.94×10 -6 These data show that the present invention has strong sensitivity, low detection limit and wide linear range.

[0028] 2. Anti-interference AFG1(10 3 ng / mL)、ZEN(10 3 ng / mL)、OTA(10 3 ng / mL) and AFB2(10 3 ng / mL) toxins were added to the prepared aptamer sensors and compared with blank samples and AFB1 (10 3 ng / mL), such as Figure 7 As shown, the SERS signals of the four interfering toxins did not change, while the electrochemical signals changed significantly, indicating that the interfering toxins had almost no effect on the aptamer sensor. This is attributed to the strong affinity and high specificity of the AFB1 aptamer, which can effectively improve the specificity of the sensor.

[0029] 3. Stability The prepared aptamer sensor was stored at 4 °C in the dark, and the Raman and electrochemical signals were measured every three days. Within 10 days, Figure 8 As shown, the RSD value of the Raman signal measurement result is 2.85%, and the RSD value of the electrochemical signal measurement result is 3.70%. The two signals did not undergo significant changes, indicating that the aptamer sensor has good stability and meets actual application requirements.

[0030] 4. Spike recovery experiment In order to verify the applicability of this method in actual samples, a spike recovery experiment was carried out in actual samples. 5g of rice and 5g of corn were selected, boiled and soaked, and 1g of sodium chloride and 25mL of methanol extract (V 甲醇 :V 水 =4:1), after 30 minutes, centrifuge at 8000 rpm / min for 15 minutes, filter the supernatant, add different concentrations of AFB1 (0.1 ng / mL, 1 ng / mL and 10 ng / mL), as shown in Table 1, the sample recoveries were 91%-117%, and RSD≤10%. These results indicate that this method is suitable for the detection of AFB1 in cereals.

[0031] Table 1 Determination results of AFB1 in actual samples The above-described embodiments are specific implementations of the present invention, which are described in relatively specific details. However, the implementations of the present invention are not limited to the above-described embodiments. Any other modifications and improvements that do not deviate from the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims

1. A method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1, characterized in that: The specific steps include: S1: Preparation of capture probe Fe3O4@Au@Ag@PAMAM@Aptamer by chemical stepwise synthesis; S2: Preparation of the signal probe AuNs@4-MBA@Ag@ssDNA by chemical stepwise synthesis; S3: PAMAM was deposited on the surface of the screen-printed electrode by electrodeposition, and the aptamer was modified by glutaraldehyde to obtain Apt / PAMAM / SPGE; the preparation process of Apt / PAMAM / SPGE was as follows: first, H2SO4 was dripped on the surface of the gold electrode, and the electrode was activated by the CV method, rinsed with deionized water, and dried naturally; then PAMAM was dripped on the surface of the gold electrode, and PAMAM was fixed to the gold electrode by the CV method, rinsed with deionized water, and dried naturally; finally, 10 μL of 10 mM aptamer standard solution was dripped on the electrode working area and incubated at 37°C; to prevent nonspecific reactions, MCH solution was incubated to block the active sites; 10 mM PBS buffer and ultrapure water were used to rinse in turn to remove excess MCH, and the mixture was dried naturally to complete the preparation of Apt / PAMAM / SPGE.

2. The method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 according to claim 1, characterized in that: The CV activated the electrode, and 50 μL of H2SO4 with a concentration of 0.5 mol / L was added on the surface of the gold electrode. The CV conditions were: scanning voltage range -1.2-1.2 V, scanning rate 100 mV / s, and scanning step length 2 m; the CV fixed PAMAM to the gold electrode, and 50 μL of PAMAM with a concentration of 0.05 mM was added on the surface of the gold electrode. The CV conditions were: scanning voltage range -0.2-1.1 V, scanning rate 25 mV / s, and scanning step length 2 mV; the concentration of the MCH solution was 0.5%; and the incubation time was 1-3 h.

3. The method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 according to claim 1, characterized in that: The preparation process of Fe3O4@Au@Ag in step S1 is as follows: 62.5 mg Fe3O4 nanoparticles and 2 mg APTES are dissolved in 15 mL ethanol, ultrasonicated for 60 min, and stirred at room temperature for 4 h; amino-functionalized Fe3O4 NPs are collected with a magnet, then rinsed with deionized water and ethanol, and dried at 70°C for 4 h; 20 mg amino-functionalized Fe3O4 NPs are dissolved in 10 mL ethanol, 20 mL AuNPs are added, and ultrasonicated at 4°C for 60 min. The formed Fe3O4@Au NPs are collected with a magnet, rinsed with deionized water, and then 300 μL 2 mM HAuCl4, 3 mL 1% sodium citrate, 600 μL 3 mM hydroquinone, and 300 mg PVP are added in sequence, and stirred at room temperature for 1-3 h to complete the gold seed growth. The formed Fe3O4@Au SP was collected with a magnet, rinsed with deionized water, dissolved in 0.2% sodium citrate, and stored at 4°C. 5 mL of 1% sodium citrate and 200 mg of PVP were added to the prepared Fe3O4@Au SP in sequence, and 300-500 μL of 30 mM AgNO3 was added dropwise at a rate of 45 s / drop at room temperature. The mixture was stirred at room temperature for 4 h. The formed Fe3O4@Au@Ag was collected with a magnet, rinsed with deionized water, dissolved in 0.2% sodium citrate, and stored at 4°C.

4. The method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 according to claim 3, characterized in that: The preparation process of the AuNPs nanoparticles is as follows: 50 mL of 2 mM HAuCl4 is heated to boiling, and then 15 mL of 1% sodium citrate is added, and the solution is stirred until the solution turns wine red, and the heat source is removed and the stirring is continued for 15 minutes to obtain a suspension of nanoparticles AuNPs, which is then rinsed with deionized water and stored at 4°C.

5. The method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 according to claim 1, characterized in that: The preparation process of Fe3O4@Au@Ag@PAMAM@Aptamer in step S1 is as follows: 1 mL of 2.5% glutaraldehyde is added dropwise to 20 mL of Fe3O4@Au@Ag mixed solution, stirred at room temperature for 2 h, 200 μL of 1 mM PAMAM is added, stirred at room temperature for 2 h, the formed Fe3O4@Au@Ag@PAMAM is collected by a magnet, rinsed with deionized water, 1 mL of 2.5% glutaraldehyde is added to the mixed solution, stirred at room temperature for 1 h, and then 12 μL of 20 mM Aptamer is added, stirred at 37°C for 1 h, and the Fe3O4@Au@Ag@PAMAM@Aptamer capture probe is collected by a magnet. To prevent nonspecific reactions, the active sites are blocked with 0.5% MCH solution.

6. The method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 according to claim 1, characterized in that: The preparation process of AuNs nanoparticles in step S2 is as follows: 5 μL of 50 mM AgNO3 and 5 mL of 5 mM HAuCl4 are added to 5 mL of 20 mM CTAB, and after stirring, 570 μL of 1% by mass ascorbic acid is added, and stirring is continued until the solution becomes transparent, and 60 μL of the prepared AuNPs is added. When the solution turns blue-brown, the mixture is stopped, and centrifuged at 4500 rpm / min for 10 min, and rinsed with deionized water to obtain AuNs, which are redispersed in 0.2% sodium citrate solution and stored at 4°C.

7. The method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 according to claim 1, characterized in that: The preparation process of AuNs@4-MBA@Ag in step S2 is as follows: add 200 μL of 5 mM 4-MBA ethanol solution to the prepared AuNs, stir continuously at room temperature for 12 h, centrifuge at 6000 rpm / min for 10 min, and rinse with deionized water to obtain AuNs@4-MBA NPs, add 3 mL of 1% sodium citrate and 90 mg of PVP in sequence, drip 200-300 μL of 30 mM AgNO3 dropwise at a speed of 15 s / drop, stir at room temperature for 2 h, centrifuge at 4500 rpm / min for 10 min, rinse with deionized water, and the obtained AuNs@4-MBA@Ag is redispersed in 0.2% sodium citrate solution and stored at 4°C.

8. The method for preparing a SERS / electrochemical dual-channel aptamer biosensor for detecting AFB1 according to claim 1, characterized in that: The preparation process of AuNs@4-MBA@Ag@ssDNA in step S2 is as follows: 25 μL of 20 mM ssDNA is dropped into 5 mL of AuNs@4-MBA@Ag complex, stirred at 4°C for 12 h, 40 μL of 1 mg / mL BSA is added, and stirred at 4°C for 6-8 h; centrifuged at 8000 rpm / min for 10 min, and rinsed with deionized water, and the prepared AuNs@4-MBA@Ag@ssDNA is stored in a PBS solution with a concentration of 0.1 mol / L and pH=7 at a temperature of -20°C.

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