Label-free functionalized SERS (Surface Enhanced Raman Scattering) aptamer sensor as well as preparation method, application and detection method thereof

Abstract

The invention provides a label-free functionalized SERS (Surface Enhanced Raman Scattering) aptamer sensor as well as a preparation method, application and a detection method. The SERS aptamer sensor is constructed by taking SiO2 as a carrier, reducing silver ions by PVP (Polyvinyl Pyrrolidone) to enable silver nanoparticles to grow on the surfaces and then combining with gold and silver dimers Au-Ag Janus NPs. Inert SiO2 is used as a carrier, after uniform and dense AgNPs grow on the surface of the carrier, an aflatoxin B1 aptamer complementary chain is modified to form a capture probe, a gold-silver nano dimer Au-Ag Janus NPs is synthesized through mediation of difunctional 2-sulfydryl-5-benzimidazole carboxylic acid MBIA, and the aflatoxin B1 aptamer is further modified to prepare a specific label-free signal probe. And the SERS aptamer sensor is formed by combining the SERS aptamer and the SERS aptamer based on a base complementary ligand principle. The SERS spectrum after the aflatoxin B1 is detected by the sensor under the excitation wavelength is collected, and a standard curve of the spectrum intensity at a characteristic Raman peak and the content of the aflatoxin B1 is established, so that the high-sensitivity rapid quantitative detection of the aflatoxin B1 in grains or food is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid food safety evaluation, and specifically relates to a label-free functionalized SERS aptamer sensor of inert silica combined with a gold-silver dimer, a preparation method, an application and a detection method, in particular an application for detecting aflatoxin B1. Background Art

[0002] Corn is the most widely grown crop in the world. It is not only an important raw material for the chemical and food industries, but also a source of feed. However, mycotoxin contamination can occur during the growth, storage and processing of corn. Aflatoxin B1 is a furanocoumarin derivative produced by Aspergillus flavus and Aspergillus parasiticus. It is one of the most widely distributed, most potent and most harmful mycotoxins. It has strong immunosuppressive, teratogenic and carcinogenic effects. China has set a maximum residue limit of 20μg / kg for aflatoxin B1 in corn.

[0003] Given the significant threat of aflatoxin B1 to human health and food safety, it is crucial to establish a reliable aflatoxin B1 detection method. Conventional aflatoxin B1 (AFB1) detection techniques mainly include enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Although these methods are widely used due to their high accuracy and excellent repeatability, they require complex sample preparation processes, expensive equipment and skilled personnel.

[0004] Surface enhanced Raman scattering (SERS) is an optical phenomenon discovered by applying laser Raman spectroscopy to surface science research. It mainly improves the Raman signal intensity of the sample to be tested through local electromagnetic enhancement caused by surface plasmon resonance (SPR), thereby realizing detection at the single molecule level. SERS technology provides highly sensitive, rapid and quantitative detection through a simple sample preparation procedure. In addition, it can collect data on molecular vibration and rotation energy levels, enabling specific detection and greatly reducing the occurrence of false positive results. Therefore, SERS has broad application prospects. However, SERS also faces the challenges of poor stability and susceptibility to external interference. Especially in the detection of fungal toxins, because the Raman signal intensity of the toxin itself is very weak and the enhancement effect with the help of SERS enhancement substrate is also poor, it is often necessary to introduce Raman beacon molecules with strong SERS signals into the SERS enhancement substrate for indirect band labeling detection. However, the introduction of beacon molecules may lead to signal instability. Correspondingly, label-free SERS detection methods for fungal toxins can avoid this problem, but most label-free detection methods do not have the specificity for detecting target substances and are easily affected by complex matrices.

[0005] In summary, it is of great significance to develop a specific label-free aflatoxin B1 detection method with high stability. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a label-free functionalized SERS aptamer sensor and its preparation method, application and detection method. The aptamer sensor described in the present invention is a SERS probe constructed based on the principle of base complementary ligands of aptamers and their complementary chains after growing silver nanoparticles (SiO2@AgNPs) on the surface of inert silica, combined with gold-silver dimers (Au-AgJanus NPs), which successfully realizes the detection of aflatoxin B1 in real corn samples and their processed products. The SERS probe described in the present invention has high sensitivity, strong specificity, and excellent stability, and is suitable for rapid batch detection of aflatoxin B1 in cereals and their products.

[0007] The present invention uses inert SiO2 as a carrier surface to grow uniform and dense AgNPs and then modify the complementary chain of aflatoxin B1 aptamer to form a capture probe, uses bifunctional 2-mercapto-5-benzimidazole carboxylic acid MBIA to mediate the synthesis of gold-silver nanodimer Au-Ag Janus NPs, and further modifies the aflatoxin B1 aptamer to prepare a specific label-free signal probe, and the two are combined based on the principle of base complementary ligands to form a SERS aptamer sensor. By collecting the SERS spectrum after the sensor detects aflatoxin B1 at the excitation wavelength, a standard curve of the spectral intensity at the characteristic Raman peak and the aflatoxin B1 content is established to achieve rapid quantitative detection of aflatoxin B1 in grain or food. The method has excellent sensitivity and specificity while achieving high stability, and is suitable for the field of food safety and analytical detection.

[0008] The present invention achieves the above technical objectives through the following technical means.

[0009] The present invention firstly provides a label-free functionalized SERS aptamer sensor. The SERS aptamer sensor is constructed by using SiO2 as a carrier to reduce silver ions with polyvinyl pyrrolidone (PVP) to grow silver nanoparticles on the surface and then combining the silver ions with gold-silver dimers (Au-Ag Janus NPs).

[0010] The present invention also provides a method for preparing the label-free functionalized SERS aptamer sensor, comprising the following steps:

[0011] S1. Preparation of SERS capture probes

[0012] S1.1 Preparation of SiO2

[0013] After mixing ethanol and NH3·H2O, stir them thoroughly until they are uniform, and slowly add a premix of ethanol and tetraethyl orthosilicate TEOS under continuous stirring. After continuous stirring at room temperature, the mixture is centrifuged and washed with ethanol, and finally dried overnight under vacuum to obtain SiO2 powder.

[0014] The volume ratio of ethanol to NH3·H2O is 8 mL:3 mL; the volume ratio of ethanol to tetraethyl orthosilicate TEOS is 8 mL:2 mL; the stirring time is 24 hours; and the centrifugal speed is 5000 rpm.

[0015] S1.2 Synthesis of SiO2@AgNPs

[0016] [Ag(NH3)2] was prepared by the following steps + Solution: Slowly add NH3·H2O to the AgNO3 solution under continuous shaking until the solution just becomes transparent, then dilute with water to obtain [Ag(NH3)2] + solution, then disperse the SiO2 powder in ethanol, followed by the rapid addition of the freshly prepared [Ag(NH3)2] + solution, the mixture was continuously stirred at room temperature and then centrifuged, washed with ethanol, and finally dried under vacuum conditions overnight to obtain SiO2@AgNPs powder. The procedure for preparing independent AgNPs solution was the same as the above steps, but without adding SiO2 powder.

[0017] The concentration of NH3·H2O is 5wt%; the volume of AgNO3 is 4mL, and the concentration is 0.4M; the total volume after dilution with water is 12mL; the ratio of SiO2 to ethanol is 0.25g:10mL; the [Ag(NH3)2] + The volume is 10 mL; the stirring time is 1 hour; and the centrifugal speed is 4000 rpm.

[0018] S1.3 Aptamer complementary strand functionalized capture probe (SiO2@AgNPs-cDNA AFB1 )

[0019] The aptamer complementary chain and TCEP were mixed in equal volumes for activation, a certain amount of SiO2@AgNPs powder obtained in step (2) was added into water to prepare a 1 mg / mL SiO2@AgNPs solution, the activated aptamer complementary chain was added into the SiO2@AgNPs solution and mixed to obtain a mixture, which was incubated at room temperature, and after the incubation was completed, the unconnected aptamer complementary chain was removed by centrifugation, and the mixture was resuspended in PBS buffer to obtain the aptamer complementary chain functionalized capture probe SiO2@AgNPs-cDNA. AFB1 Solution, save.

[0020] Wherein, the incubation time is 12h; the nucleotide sequence of the aptamer complementary chain is:

[0021] 5'-SH-CAGAGAGACAACACGTGCCCAAC-3', modified with a thiol group at the 5' end.

[0022] The pH value of the PBS buffer is 7.2-7.4, and the PBS buffer contains 2mM H2PO4, 8mM Na2HPO4, 136mM NaCl, and 2.6mM KCl.

[0023] The method for aptamer activation is as follows: TCEP solution and aptamer complementary chain solution are mixed in a volume ratio of 1:1, and reacted for 40 minutes to activate the thiol-modified aptamer complementary chain; the molar ratio of the aptamer complementary chain to TCEP is 1:100. The concentration of the aptamer complementary chain is 100 μM, and the volume used for mixing with the SiO2@AgNPs solution is 10 μL.

[0024] S2. Preparation of signal probe

[0025] S2.1 Synthesis of AuNPs

[0026] HAuCl4·4H2O solution was introduced into deionized water and heated to boiling, then trisodium citrate solution was quickly added while vigorously stirring, and the mixture was continuously boiled until the color remained unchanged to obtain an AuNPs solution, after which the previous AuNPs were cooled to room temperature for use.

[0027] The concentration of HAuCl4·4H2O is 1wt%, and the volume ratio of HAuCl4·4H2O to deionized water is 0.5mL:49.5mL; the concentration of trisodium citrate solution is 1wt%, and the volume is 1mL.

[0028] S2.2 Preparation of Au-Ag Janus NPs

[0029] The AuNPs solution obtained in step S2.1 was mixed with deionized water until uniform, and 2-mercapto-5-benzimidazolecarboxylic acid MBIA solution was added. The mixed solution was placed in a 60°C water bath environment, then taken out, and hydroquinone HQ solution was added after it was cooled to room temperature. Subsequently, different volumes of AgNO3 solution were slowly added dropwise under vigorous stirring. The obtained solution was centrifuged and redissolved in water after being left at room temperature overnight.

[0030] Among them, the volume ratio of the AuNPs solution to deionized water is 1mL:9mL; the volume of the MBIA solution is 200μL, and the maintenance time of the solution in the water bath environment is 2 hours; the volume of the HQ solution is 200μL; the volume of the AgNO3 solution is 300-1800μL, preferably, the volume of the AgNO3 solution is 600μL, and the centrifugal speed is 6000rpm; the re-dissolved volume of the solution after centrifugation is 2mL.

[0031] S2.3 Aptamer-functionalized signal probe Au-Ag Janus NPs-apt AFB1 Preparation

[0032] The activated aptamer Apt AFB1 Add the mixture to the Au-Ag Janus NPs solution and mix well to obtain a mixture, incubate at room temperature, centrifuge to remove unconnected aptamers after incubation, and resuspend in PBS buffer to obtain the aptamer functionalized signal probe Au-Ag Janus NPs-apt AFB1 Solution, save.

[0033] Wherein, the incubation time is 12h; the aptamer Apt AFB1 The nucleotide sequence is:

[0034] 5'-SH-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3', with thiol modification at the 5' end.

[0035] The PBS buffer conditions are the same as step S1.3.

[0036] The method and concentration of aptamer activation are the same as those of the aptamer complementary chain in step S1.3. The volume used for mixing with the Au-AgJanus NPs solution is 5 μL.

[0037] S3. Preparation of SERS aptasensor

[0038] The capture probe obtained in step S1 and the signal probe obtained in step S2 are incubated for different times at different volume ratios. After incubation, the solution is centrifuged and re-dissolved to obtain the desired SERS aptamer sensor SiO2@AgNPs-Au-Ag Janus NPs.

[0039] The signal probe is fixed on the capture probe surface through the base complementary pairing principle, and the unbound signal probe is removed by centrifugation.

[0040] The volume ratio of the capture probe to the signal probe is 1:1 to 1:5, preferably, the volume ratio is 1:4; the incubation time is 0 to 3 hours, preferably, the incubation time is 2 hours.

[0041] The present invention also provides application of the label-free functionalized SERS aptamer sensor in detecting aflatoxin B1.

[0042] The present invention also provides a method for detecting aflatoxin B1 based on the label-free functionalized SERS aptamer sensor, comprising the following steps:

[0043] (1) The label-free functionalized SERS aptamer sensor solution and the aflatoxin B1 standard solution were mixed in equal volumes, the reaction was captured and re-dissolved by centrifugation, and the Raman spectrum was collected.

[0044] The volume of the aflatoxin B1 standard solution is 100 μL, the concentration is 0.0001-100 ng / mL, and the capture time is 2 h.

[0045] (2) Establish a standard curve based on the relationship between the concentration of aflatoxin B1 (AFB1) standard solution and the characteristic Raman peak. Preferably, the standard curve is y=-3286.44x+11927.26, R 2 =0.990, and the detection limit of AFB1 is 0.00134 μg / kg.

[0046] (3) Detecting the content of aflatoxin B1 in the sample to be tested: mixing the sample solution to be tested with the above-mentioned label-free functionalized SERS aptamer sensor, collecting Raman spectra according to the method of step (1), and calculating the content of aflatoxin B1 in the sample to be tested according to the standard curve of step (2).

[0047] The samples to be tested are corn flour naturally contaminated with aflatoxin B1, spiked corn flour, and spiked corn oil.

[0048] The above method detects the concentration of aflatoxin B1 in the sample solution, and further calculates the content of aflatoxin B1 in the sample to be tested according to the measured concentration. The calculation formula of the content of aflatoxin B1 in the sample to be tested in the present invention is as follows:

[0049]

[0050] X: The content of aflatoxin B1 in the sample to be tested, in micrograms per kilogram (μg / kg),

[0051] ρ: the concentration of aflatoxin B1 in the sample solution, in nanograms per milliliter (ng / mL),

[0052] V: the volume of the sample solution to be tested, in milliliters (mL).

[0053] m: The weight of the sample to be tested, in grams (g).

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] (1) The present invention uses SiO2 with large surface area, high stability and biocompatibility as a carrier, so that AgNPs grow densely and uniformly on its surface, improving the easy oxidation of AgNPs and the easy aggregation and low uniformity caused by their high surface energy. At the same time, the dense distribution of AgNPs enables it to generate more hot spots, promoting the generation of higher intensity SERS signals. Au-Ag Jnaus NPs with inherent Raman signals replace beacon molecules to generate Raman signals, avoiding the negative impact of the introduction of beacon molecules on the stability of the system and reducing its sensitivity to external conditions.

[0056] (2) The complementary chain of aflatoxin B1 aptamer and the aflatoxin B1 aptamer were introduced into SiO2@AgNPs and Ag-Ag Janus NPs, respectively, so that the two could combine to form a SERS aptamer sensor based on the complementary base pairing principle between nucleic acids, thereby realizing label-free specific SERS detection of aflatoxin B1. The combination of the two has the dual functions of improving the weak stability of Au-AgJanus NPs under acidic conditions and significantly enhancing the SERS signal intensity. This expands the detection range of SERS probes for aflatoxin B1 and expands its application range.

[0057] (3) The probe and detection method of the present invention can realize the specific rapid detection of aflatoxin B1. It is only necessary to mix the prepared SERS probe with the sample to be tested in equal volumes. After the incubation is completed, the sample is centrifuged and re-dissolved and the signal is collected. The correlation between the intensity of the Raman characteristic peak displayed by the aptamer sensor and the concentration of the corresponding target is established, and the detection can be completed quickly and accurate detection results can be obtained. The detection process is completed within 3 minutes, and the detection limit of AFB1 can reach 0.00134μg / kg. For actual samples, the detection results of the method of the present invention are consistent with the traditional HPLC method, with good stability and RSD (n=5) of 0.36%-1.56%. The preparation and detection process of the present invention is simple and efficient, and realizes high-sensitivity and rapid detection of aflatoxin B1. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a schematic diagram of the preparation process of the label-free functionalized SERS aptamer sensor of the present invention and the method for quantitatively detecting aflatoxin B1.

[0059] Figure 2is the SiO2 prepared in Example 1 ( Figure 2 A) and SiO2@AgNPs( Figure 2 B- Figure 2 C) morphological features and actual picture.

[0060] Figure 3 Characterization diagram for verifying the successful preparation of SiO2@AgNPs in Example 1: XRD: Figure 3 A; XPS: Figure 3 B- Figure 3 C.

[0061] Figure 4 For SiO2( Figure 4 A) and SiO2@AgNPs( Figure 4 B) with a concentration of 4.5×10 -4 After incubation with 4-MBA, the peak of M was 1589cm -1 Raman signal intensity of 30 randomly selected points.

[0062] Figure 5 AgNPs, SiO2, SiO2@AgNPs and 4.5×10 -4 Raman spectra of M after incubation with 4-MBA.

[0063] Figure 6 is the zeta potential of AgNPs, SiO2, and SiO2@AgNPs.

[0064] Figure 7 To capture the changes in the UV-visible absorption spectrum during the synthesis of the probe.

[0065] Figure 8 The morphological characteristics and physical pictures of AuNPs prepared in Example 1 ( Figure 8 A) and the particle size distribution of AuNPs ( Figure 8 B).

[0066] Fig. 9 The Au-Ag Janus prepared in Example 1 (1) NPs( Fig. 9 A) Au-Ag Janus (2) NPs( Fig. 9 B) Au-Ag Janus (3) NPs( Fig. 9 C) and Au-Ag Janus (4) NPs( Fig. 9 D) Silver island length distribution diagram and its corresponding length at 1280cm -1 The Raman signal intensity at Fig. 9 E).

[0067] Fig.10 To verify the successful preparation of Au-Ag Janus NPs in Example 1, the morphological characteristics and physical images of Au-Ag Janus NPs ( Fig.10 A), UV-visible absorption spectra of AuNPs and Au-Ag Janus NPs ( Fig.10 B), STEM element content of Au-AgJanus NPs ( Fig.10 C).

[0068] Fig.11 The Au-Ag Janus NPs at different pH ( Fig.11 A), temperature, storage time ( Fig.11 B) Raman signal intensity diagram under different conditions.

[0069] Fig.12 This is a diagram of the zeta potential change during the preparation of the signal probe.

[0070] Fig.13 The graph shows the change in Raman signal intensity after the capture probe and signal probe were incubated at different volume ratios.

[0071] Fig.14 The graph shows the change in Raman signal intensity after the capture probe and the signal probe are incubated for different time periods at the optimal volume ratio.

[0072] Fig.15 The morphological characteristics of the aptamer sensor ( Fig.15 A- Fig.15 B) and STEM element distribution map ( Fig.15 C).

[0073] Fig.16 UV-visible absorption spectra of SiO2@AgNPs, Au-Ag Janus NPs, and SERS aptamer sensors ( Fig.16 A) and STEM element content of SERS aptasensor ( Fig.16 B).

[0074] Fig.17 Raman spectra of SiO2@AgNPs, Au-Ag Janus NPs, and SERS aptamer sensors.

[0075] Fig.18 This is a Raman spectrum graph collected for the SERS aptamer sensor prepared in Example 1 to detect the AFB1 standard.

[0076] Fig.19 The SERS aptamer sensor prepared in Example 1 detected AFB1 at 1280 cm -1The linear fitting curve is plotted between the Raman signal intensity of the characteristic peak at and the lg value of the corresponding AFB1 standard solution concentration.

[0077] Fig. 20 The stability verification results of the SERS aptamer sensor prepared in Example 1 ( Fig. 20 A) and the repeatability verification results of the SERS aptamer sensor prepared in Example 1 ( Fig. 20 B- Fig. 20 C).

[0078] Fig.21 This is the specific verification result of the SERS aptamer sensor prepared in Example 1. DETAILED DESCRIPTION

[0079] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following is a detailed description in conjunction with specific embodiments, but the following embodiments do not limit the scope of protection of the present invention. In the embodiments of the present invention, all that is not described in detail is completed using conventional experimental methods, and all that is not described in detail in the processes involved in the embodiments is understandable and easily implemented by those skilled in the art based on the product manual or basic knowledge in the art, and therefore will not be described in detail.

[0080] Example 1: Preparation method of SERS aptamer sensor for detecting aflatoxin B1

[0081] use Method (Physical and chemical method for synthesizing monodisperse silica particles) The newly prepared [Ag(NH3)2] + ion solution is introduced into the silica solution. Due to the negatively charged silanol groups, [Ag(NH3)2] + The ions easily attach to the surface of the silica nanospheres through electrostatic interactions. Then, polyvinyl pyrrolidone (PVP) is introduced to reduce [Ag(NH3)2] + ions, synthesize SiO2@AgNPs and stabilize AgNPs. Using 2-mercapto-5-benzimidazolecarboxylic acid MBIA as a ligand and hydroquinone HQ as a reducing agent, silver islands were guided to grow on gold nanoparticles synthesized by the sodium citrate method. The resulting Au-AgJanusNPs showed an intrinsic SERS signal. SiO2@AgNPs and Au-Ag Janus NPs were modified with aflatoxin B1 aptamer complementary chain and aflatoxin B1 aptamer, respectively. Under optimal conditions, aptamer sensors were formed based on complementary base pairing, showing enhanced SERS signals. When aflatoxin B1 (AFB1) is present, the aptamer Apt AFB1The Au-Ag Janus NPs preferentially bind to the SiO2@AgNPs, resulting in the separation of the Au-Ag Janus NPs from the SiO2@AgNPs and the weakening of the signal, thereby enabling the construction of a competitive SERS aptamer sensor for the quantitative detection of aflatoxin B1 (AFB1). Figure 1 shown.

[0082] The specific steps are as follows:

[0083] S1. Capture probe SiO2@AgNPs-cDNA AFB1 Preparation

[0084] (1) Preparation of SiO2:

[0085] After 8 mL of ethanol and 3 mL of NH3·H2O were uniformly mixed, a mixed solution of 8 mL of ethanol and 2 mL of LTEOS was slowly added dropwise while stirring. The mixture was stirred at room temperature for 24 hours, centrifuged at 5000 rpm, washed with ethanol 6 times, and finally dried in a vacuum environment at 60°C overnight to obtain SiO2 powder.

[0086] (2) Preparation of SiO2@AgNPs

[0087] Under continuous stirring, 5 wt% NH3·H2O was slowly added dropwise to 4 mL of 0.4 M AgNO3 solution until the solution just became transparent, and then diluted with water to a total volume of 12 mL to prepare [Ag(NH3)2] + Solution. Disperse 0.25 g of silica powder in 10 mL of ethanol. Then, quickly add 10 mL of freshly prepared [Ag(NH3)2] + The solution was stirred continuously at room temperature for 1 hour. It was then centrifuged at 4000 rpm, washed with ethanol for 6 times, and then dried under vacuum at 60 °C overnight to obtain SiO2@AgNPs powder. The procedure for preparing independent AgNPs solution was the same as above, but without adding SiO2 powder.

[0088] The morphological characteristics and physical properties of SiO2 obtained in this embodiment are as follows Figure 2 As shown in Figure A, SiO2 appears as regular spherical nanoparticles, with a white powder appearance and a particle size of about 750nm. Figure 2 B- Figure 2 As shown in C, dense AgNPs grow on the surface of SiO2 nanospheres, and the appearance is brown powder with a particle size of about 868nm.

[0089] XRD characterization of SiO2 and SiO2@AgNPs Figure 3As shown in Figure A, it shows that SiO2 has an amorphous diffraction peak of 2θ at 24°. SiO2@AgNPs shows new diffraction peaks at 38.15, 44.26, 64.48 and 77.52, corresponding to the (111), (200), (220), and (311) crystal phases of silver, which indicates that crystalline AgNPs have been formed. The XPS total spectrum of SiO2@AgNPs is shown in Figure Figure 3 As shown in Figure B, the appearance of the characteristic peaks of C 1s and N1s indicates the presence of PVP on the surface of SiO2@AgNPs, while the presence of the characteristic peaks of O1s, Si2p and Ag 3d confirms the presence of silicon, oxygen and silver materials in SiO2@AgNPs, respectively. The specific XPS spectrum corresponding to Ag3d is shown in Figure 2. Figure 3 C, XPS analysis of Ag 3d reveals two peaks at 368.2 and 374.2 eV with a spin-orbit spacing of 6 eV, corresponding to Ag 3d 5 / 2 and Ag 3d 3 / 2 The binding energy confirms the existence of zero-valent Ag. The above characterization results provide a reliable basis for the successful preparation of SiO2@AgNPs.

[0090] SiO2 and SiO2@AgNPs were mixed with 4.5×10 -4 After incubation with 4-MBA, the peak of M was 1589cm -1 The SERS intensities of 30 randomly collected points are as follows: Figure 4 A and Figure 4 As shown in Figure 2B, AgNPs showed an RSD of 20.40%, while SiO2@AgNPs showed a lower RSD of 8.16%, demonstrating the improvement of the uniformity of AgNPs by SiO2 as a carrier.

[0091] AgNPs, SiO2, and SiO2@AgNPs were respectively -4 The Raman spectrum of M after incubation with 4-MBA is shown in Figure 5 As shown in Figure 2, silica exhibited no discernible SERS enhancement. Even at relatively low concentrations, AgNPs bound to silica showed comparable signal intensities to higher concentrations of individual AgNPs, confirming that the dense distribution of AgNPs on SiO2 facilitated the generation of hotspots that led to signal enhancement.

[0092] The zeta potential of AgNPs, SiO2, and SiO2@AgNPs is shown in Figure 6As shown in the figure, according to the principle that the higher the absolute value of the zeta potential, the better the physical stability, the average potential of AgNPs is -18.46mV and the average potential of SiO2 is -66.2mV, indicating that the absolute value is very high, which is consistent with its excellent stability. The average absolute value of the potential of SiO2@AgNPs is slightly lower (-60.78mV), but the value is still very high, confirming the improvement of the stability of AgNPs by SiO2.

[0093] (3) Aptamer complementary chain functionalized capture probe SiO2@AgNPs-cDNA AFB1 Preparation

[0094] The thiol-modified aptamer complementary chain and TCEP were mixed at a molar ratio of 1:100 and incubated for 40 minutes to activate the thiol groups at the ends of the aptamer complementary chain. Then, 10 μL of the activated aflatoxin B1 aptamer complementary chain with a concentration of 100 μM was further added to 1 mL of the fully sonicated SiO2@AgNPs solution with a concentration of 1 mg / mL and incubated with shaking for 12 hours. After centrifugation, the solution was resuspended in PBS buffer.

[0095] SiO2@AgNPs-cDNA AFB1 The changes of UV-visible absorption spectrum during the preparation process are as follows: Figure 7 As shown, SiO2 did not show an obvious UV-visible absorption peak, while SiO2@AgNPs showed an absorption peak at 408 nm, which was attributed to AgNPs. After incubation with the aptamer complementary chain, a new peak appeared at 264 nm, indicating the successful coupling between the nanoparticles and the aptamer complementary chain.

[0096] S2. Signal probe Au-Ag Jnaus NPs-apt AFB1 Preparation

[0097] (1) Preparation of AuNPs

[0098] 0.5 mL of 1 wt% HAuCl4·4H2O was introduced into 49.5 mL of water and heated to boiling. Then, 1 mL of 1 wt% trisodium citrate solution was quickly added while stirring vigorously. The mixture was boiled continuously until the color remained unchanged. Then, the mixture was cooled to room temperature.

[0099] The morphological characteristics and physical properties of AuNPs Figure 8 As shown in A, it is a nearly spherical nanoparticle uniformly dispersed in the solution and the solution appears purple-red. The particle size distribution of AuNPs is shown in Figure 8 As shown in B, it is calculated to be approximately 38.96nm.

[0100] (2) Preparation of Au-Ag Janus NPs

[0101] 1 mL of AuNPs was dissolved in 9 mL of deionized water, mixed well, and then 200 μL of MBIA was added and placed in a water bath at 60 ° C for 2 hours. When cooled to room temperature, 200 μL of HQ was introduced. After the solution was evenly mixed, 300, 600, 1200, and 1800 μL of AgNO3 solution were slowly added dropwise under vigorous stirring. The resulting solution was kept at room temperature overnight, centrifuged at 6000 rpm, and then redissolved in 2 mL of water. The resulting nanoparticles were named Au-Ag Janus (1) NPs, Au-AgJanus (2) NPs, Au-Ag Janus (3) NPs and Au-Ag Janus (4) NPs.

[0102] The volume of AgNO3 added in step (2) was optimized to obtain the Au-Ag Janus (1) NPs, Au-AgJanus (2) NPs, Au-Ag Janus (3) NPs and Au-Ag Janus (4) The Ag island length distribution diagrams of NPs are shown in Fig. 9 A- Fig. 9 As shown in D, the length of the silver island increases with the addition of AgNO3, which are 30.56, 42.20, 52.65 and 80.62 nm respectively. The changes in the Raman signal intensity of the four Au-Ag Janus NPs are shown in Fig. 9 As shown in E, the signal begins to increase as the length of the Ag island increases. (2) NPs obtained the highest SERS signal. In addition, the oversized Ag islands caused the structure of the dimerized nanoparticles to become irregular, resulting in decreased signal reproducibility and stability. Therefore, 600 μL was selected as the optimal dosage of AgNO3.

[0103] In this embodiment, Au-Ag Janus (2) The morphology and physical properties of NPs Fig.10 As shown in A, it shows a unique anisotropic structure, indicating the successful growth of Ag islands on the side of the Au core-shell. At the same time, the solution color turns brown. (2) The UV-visible absorption spectra of NPs are shown in Fig.10 As shown in B, AuNPs show a strong UV-visible absorption peak at 526 nm. (2)NPs, two peaks were observed at 400nm and 800nm, corresponding to the lateral absorption of Ag NPs and the longitudinal absorption of Au-Ag Janus NPs, respectively. Meanwhile, the absorption peak of AuNPs blue-shifted to 513nm. (2) The overall STEM element content of NPs is shown in Fig.10 C, indicating that Ag and Au elements exist simultaneously. The above characterizations all verify the successful preparation of Au-Ag Janus NPs.

[0104] Au-Ag Janus (2) The stability of NPs under different pH conditions Fig.11 As shown in A, in the pH range of 7-11, the nanoparticles have a -1 The system aggregated at pH 6.5 and showed a significant decrease in signal intensity, which further decreased with decreasing pH. This indicates that Au-AgJanus nanoparticles have excellent stability under neutral and alkaline conditions, but are relatively weak under acidic conditions. (2) The stability of NPs at different temperatures and storage times is shown in Figure 2. Fig.11 As shown in Figure B, this figure shows that Au-AgJanus (2) The SERS signal intensity of NPs did not change significantly at different temperatures or storage times, demonstrating their good environmental adaptability.

[0105] (3) Aptamer-functionalized signal probe Au-Ag Janus NPs-apt AFB1

[0106] The thiol-modified aptamer and TCEP were mixed at a molar ratio of 1:100 and incubated for 40 minutes to activate the thiol groups at the ends of the aptamer. Then, 5 μL of the activated aflatoxin B1 aptamer complementary chain at a concentration of 100 μM was further added to 1 mL of the fully sonicated SiO2@AgNPs solution at a concentration of 1 mg / mL and incubated with shaking for 12 hours. After centrifugation, the solution was resuspended in PBS buffer.

[0107] Au-Ag Janus NPs-apt AFB1 The zeta potential changes during the preparation process are as follows: Fig.12 As shown, the zeta potential of gold-MBIA is -33.12 mV, while that of Au-Ag Janus NPs decreases to -22.76 mV. After the nanoparticles are coupled with the aptamer, the negative potential increases to -25.91 mV due to the negative charge provided by the phosphate group in the aptamer.

[0108] S3. Preparation of SERS aptamer sensor (SiO2@AgNPs-cDNA-apt-Au-Ag Janus NPs)

[0109] The prepared capture probe (SiO2@AgNPs-cDNA AFB1 ) and signal probe (Au-Ag Jnaus NPs-apt AFB1 ) were incubated at a volume ratio of 1:1, 1:2, 1:3, 1:4, and 1:5, respectively. After determining the preferred volume ratio of the two, the mixed solution was allowed to stand for 0, 1, 1.5, 2, 2.5, and 3 hours at the preferred volume ratio, and the capture probe and the signal probe were coupled according to the base complementary pairing principle of the aptamer and the corresponding complementary chain. Subsequently, the mixed solution was centrifuged at 2500 rpm and then redissolved to prepare a label-free functionalized SERS aptamer sensor.

[0110] The obtained SERS aptamer sensor not only improved the weak stability of Au-Ag JanusNPs under acidic conditions, but also significantly increased the response intensity of the Raman signal. -1 The characteristic peak intensity at is taken as the response signal intensity. In the absence of target AFB1, the characteristic peak of the aptamer sensor has a strong Raman response. Since aflatoxin B1 is superior to its aptamer complementary chain in binding to the aptamer, the sensor assembly dissociates and the signal probe is released from the capture probe SiO2@AgNPs. Therefore, the Raman signal intensity from the capture probe is weakened.

[0111] The volume ratio of the capture probe to the signal probe in step S3 is optimized, such as Fig.13 As shown in the figure, as the ratio of the two probes increases from 1:1 to 1:4, the SERS signal intensity of the system gradually increases and reaches its maximum value. However, when the ratio continues to increase to 1:5, the signal intensity no longer increases. This is because the ratio of 1:4 achieves saturated binding of Au-Ag Janus NPs on SiO2@AgNPs. When more Au-Ag Janus NPs are added, no further coupling occurs. Therefore, 1:4 is selected as the optimal binding ratio.

[0112] The incubation time of the capture probe and the signal probe in step S3 is optimized, such as Fig.14 As shown in the figure, with the increase of incubation time, the signal intensity of the system gradually decreased, which was due to the superiority of aflatoxin B1 over its aptamer complementary chain and the aptamer ensemble, resulting in the reduction of Au-Ag Janus NPs on SiO2@AgNPs. After 2 hours of incubation, the SERS signal intensity stabilized and reached the minimum. Therefore, the ideal reaction duration was determined to be 2 hours.

[0113] The morphological characteristics of aptasensors are as follows Fig.15 A- Fig.15 As shown in B, it shows that Au-Ag Janus NPs are attached to the surface of SiO2@AgNPs. Fig.15 As shown in C(ad), the STEM element distribution map proves the simultaneous existence of Si, O, Ag and Au elements in the composite component, further verifying the formation of SiO2@AgNPs-Au-Ag Janus NPs assembly.

[0114] UV-visible absorption spectra of SiO2@AgNPs, Au-Ag Janus NPs, and SERS aptamer sensors Fig.16 As shown in A, the assembly retains the UV-visible absorption peak characteristics of Au-Ag Janus NPs, while the peak of AgNPs is enhanced and the peak of AuNPs is weakened. This is due to the higher content of AgNPs in the aptamer sensor and the lower relative content of AuNPs. This explanation is Fig.16 B and Fig.10 The comparison of C can further confirm this.

[0115] Raman spectra of SiO2@AgNPs, Au-Ag Janus NPs, and SERS aptamer sensors Fig.17 As shown, SiO2@AgNPs itself does not have an obvious SERS signal, while Au-Ag Janus NPs exhibit an intrinsic Raman signal. The SERS aptasensor exhibits an obvious SERS enhancement effect based on the signal intensity of Au-Ag Janus NPs.

[0116] Embodiment 2:

[0117] The SERS aptamer sensor prepared in Example 1 was applied to the quantitative detection of aflatoxin B1.

[0118] (1) A standard solution with a concentration gradient of 0.0001, 0.001, 0.01, 0.1, 1, 10, 50, and 100 ng / mL was prepared using aflatoxin B1 standard. The above-mentioned SERS aptamer sensor was mixed with the standard solution in equal volumes. After capturing for 2 hours, the free signal probe was removed by centrifugation at 2000 rpm. A fully automatic microscopic Raman spectrometer was used to collect and process SERS spectral data. First, a 5× low-magnification objective was used to roughly adjust the droplet sample to a clear focus, and then a 10× high-magnification objective was switched to obtain a clear microscopic image by adjusting the field brightness and focus.

[0119] The acquisition parameters were set as follows: the excitation wavelength was 638 nm, the excitation power was 100 mW, and the integration time was 10 s. Each sample was acquired 5 times.

[0120] (2) Data processing and analysis: Reliable quantitative and qualitative analysis of the original SERS spectra can effectively improve the accuracy of the detection method. The original spectra were preprocessed by chemometric methods using LabSpec6 software to eliminate background drift caused by fluorescence absorption; the spectrum was baseline calibrated by background fitting (polynomial) to remove background interference, and the parameters were set to degree = 9, maximum points = 82; the smoothing algorithm selected Polynomial polynomial, and the parameters were set to factor = 10, Degree = 3, Size = 12.

[0121] The Raman spectra collected by using the SERS aptamer sensor to detect the AFB1 standard are as follows: Fig.18 As shown in the figure, it can be seen that the SERS aptamer sensor has a wavelength of 1280 cm -1 The intensity of the Raman characteristic peak at decreases with the increase of AFB1 concentration, which is consistent with the competitive principle of the sensor.

[0122] The AFB1 detected by SERS aptamer sensor is at 1280 cm -1 The linear fitting curve drawn by the Raman signal intensity of the characteristic peak at 1000 nm and the lg value of the corresponding AFB1 standard solution concentration is shown in the figure. Fig.19 As shown in the figure, it can be seen that when the concentration range of AFB1 is 0.001-100 ng / mL, the curve has good linear characteristics (y=-3286.44x+11927.26, R 2 =0.990). According to the definition of the aflatoxin B1 concentration corresponding to a 10% decrease in the SERS signal intensity of the aptamer sensor relative to the blank sample, the detection limit of this scheme for AFB1 was calculated to be 0.00134 μg / kg.

[0123] The SERS signals of the aptamer sensor prepared in Example 1 at different pH values ​​(4, 5, 6, 7, 8, 9, 10, 11), temperatures (20, 35, 45, 60° C.) and storage times (0, 7, 14 days) were compared to verify the stability of the sensor:

[0124] like Fig. 20 As shown in Figure A, the sensor exhibited similar SERS signal intensity in the pH range of 4-11, the temperature of 20-60°C, and the storage time of 0-14 days, which showed that it had excellent stability and improved the weak stability of Au-AgJanus NPs under acidic conditions, making the sensor have a wider range of application environments.

[0125] The 36 points of the SERS aptamer sensor incubated with 50 ng / mL aflatoxin B1 standard solution at 1280 cm were randomly collected. -1 The SERS spectra at 400 nm were taken to verify the reproducibility of the detection method.

[0126] like Fig. 20 As shown in BC, the Raman characteristic peaks of MBIA show similar intensities and are at 1280 cm -1 The RSD value of the signal intensity at 400 nm was 5.97%, indicating that the sensor had satisfactory reproducibility.

[0127] Standard solutions of different types of toxins were used to react with the SERS aptamer sensor prepared in Example 1 to verify the specificity of the detection method:

[0128] Other common toxins in corn: zearalenone (ZEN), fumonisins (FB1), ochratoxin (OTA), deoxynivalenol (DON) (all at 10 ng / mL) were selected as interfering substances. They were incubated with the aptamer sensor established under optimal conditions using the same method as the detection of aflatoxin B1 (AFB1) (at a concentration of 0.1 ng / mL). The changes in SERS intensity were then compared. At the same time, a blank solution was prepared as a control.

[0129] like Fig.21 As shown, although the concentration of interfering toxin was 100 times that of AFB1, Fig.21 It can be seen that other toxins did not cause any obvious changes in the Raman signal, however, with the introduction of AFB1, the signal showed a significant decrease, which indicates that the aptamer sensor prepared in Example 1 has excellent specificity for aflatoxin B1.

[0130] The label-free functionalized SERS aptamer sensor of the present invention detects aflatoxin B1 in real corn samples:

[0131] In order to verify the practical operability of this method in real corn samples (including corn flour and corn oil), the SERS detection method was used to detect the aflatoxin B1 content in corn flour naturally contaminated with aflatoxin B1 and spiked samples (corn flour or corn oil not contaminated with aflatoxin B1 mixed with standard substances).

[0132] The specific method is:

[0133] (1) Prepare sample solution (sample solution to be tested):

[0134] Equal volumes of aflatoxin B1 standard solutions of different concentrations were added to 1 g of corn flour to simulate contaminated corn tissue (this step is not required for naturally contaminated corn flour). The sample was placed at room temperature for 2 hours, then 10 ml of methanol (70%, w / v) and 0.4 g of sodium chloride were added, vortexed for 20 minutes, and then centrifuged at 6000 rpm for 10 minutes. The resulting supernatant can be used for SERS detection. The pretreatment steps of corn oil are the same as corn flour.

[0135] (2) The SERS aptamer sensor prepared in Example 1 was immersed in a standard solution of aflatoxin B1 (concentration of 0.0001-100 ng / mL). After 2 h of capture reaction, Raman spectra were collected. The Raman spectra were compared with the concentration of the standard solution of aflatoxin B1 (AFB1) and the aptamer sensor at 1280 cm -1 The standard curve was established by comparing the peak intensities of

[0136] (3) The SERS aptamer prepared in Example 1 was incubated with the prepared sample solution and then centrifuged for redissolution to collect Raman spectra. The acquisition parameter settings and data processing and analysis methods were the same as the data processing method for establishing the standard curve in Example 2. According to the intensity of the acquired spectrum, the established standard curve was called to detect the concentration of aflatoxin B1 in the sample solution.

[0137] The content of aflatoxin B1 in the sample to be tested is further calculated according to the measured concentration, and in the present invention, it is calculated by the following formula:

[0138]

[0139] X: The content of aflatoxin B1 in the sample to be tested, in micrograms per kilogram (μg / kg)

[0140] ρ: Concentration of aflatoxin B1 in the sample solution, in nanograms per milliliter (ng / mL)

[0141] V: the volume of the sample solution, in milliliters (mL)

[0142] m: The weight of the sample to be tested, in grams (g)

[0143] The detection value and recovery rate determined by the method of the present invention are compared with those of the liquid chromatography method as shown in Table 1. The recovery rate of AFB1 determined by the method of the present invention is between 88.70% and 114.04%, which is close to the result of the standard HPLC method and is in the range of 80%-120%, verifying the reliability of the detection method of the present invention.

[0144] Table 1. Detection results of aflatoxin B1 in actual samples by this method and HPLC

[0145]

[0146] In summary, the label-free functionalized SERS aptamer sensor designed by the present invention is a SERS aptamer sensor based on SiO2@AgNPs-Au-Ag Janus NPs. This is a portable SERS sensor with good stability and environmental friendliness, and can quickly detect the content of aflatoxin B1 in corn products. By utilizing the specific binding of the aptamer and the corresponding complementary chain based on base complementary pairing and the affinity of the target aflatoxin B1 for the aptamer that is superior to the complementary chain, a negative correlation specific detection result that eliminates the interference of other substances and fungal toxins in complex samples can be obtained. Through subsequent calculations, the peak value at the characteristic Raman shift of MBIA in the sensor is correlated with the content of aflatoxin B1, without the need for cumbersome pre-treatment steps, and is expected to replace traditional liquid phase and liquid phase-mass spectrometry to detect aflatoxin B1 in food and achieve highly sensitive quantitative detection.

[0147] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail in this specification with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A label-free functionalized SERS aptamer sensor, characterized in that: The SERS aptamer sensor is constructed by using SiO2 as a carrier to reduce silver ions with polyvinyl pyrrolidone (PVP) to grow silver nanoparticles on the surface and then combining them with gold-silver dimers Au-Ag Janus NPs.

2. The method for preparing the label-free functionalized SERS aptamer sensor according to claim 1, characterized in that: The following steps are involved: S1. Preparation of SERS capture probes S1.1 Preparation of SiO2 After mixing ethanol and NH3·H2O, stir them thoroughly until they are uniform, and then add a premix of ethanol and tetraethyl orthosilicate TEOS dropwise. After continuously stirring the mixture, centrifuge it, wash it with ethanol, and finally dry it to obtain SiO2 powder. S1.2 Synthesis of SiO2@AgNPs Add NH3·H2O dropwise to the AgNO3 solution until the solution just becomes transparent, and dilute with water to obtain [Ag(NH3)2] + Solution; dispersing the SiO2 powder obtained in step S1.1 in ethanol, adding the [Ag(NH3)2] + The solution was stirred and centrifuged, washed with ethanol, and dried overnight to obtain SiO2@AgNPs powder; S1.3 Aptamer complementary strand functionalized capture probe SiO2@AgNPs-cDNA AFB1 Preparation After the aptamer complementary chain is activated by mixing equal volumes of TCEP, it is added to the SiO2@AgNPs solution obtained in step S1.2 and mixed evenly. After incubation at room temperature, the unconnected aptamer complementary chain is removed by centrifugation and resuspended in PBS buffer to obtain the aptamer complementary chain functionalized capture probe SiO2@AgNPs-cDNA. AFB1 Solution; S2. Preparation of signal probe S2.1 Synthesis of AuNPs HAuCl4·4H2O solution was introduced into deionized water and heated to boiling, trisodium citrate solution was added, and the mixture was continuously boiled until the color remained unchanged to obtain AuNPs solution; S2.2 Preparation of Au-Ag Janus NPs The AuNPs solution obtained in step S2.1 was mixed with deionized water until uniform, and 2-mercapto-5-benzimidazole carboxylic acid MBIA solution was added. The mixed solution was placed in a water bath environment, and after being taken out and cooled to room temperature, the hydroquinone HQ solution was added, and the AgNO3 solution was slowly added dropwise with stirring. The obtained solution was centrifuged and redissolved in water after being left at room temperature overnight; S2.3 Aptamer-functionalized signal probe Au-Ag Janus NPs-apt AFB1 Synthesis Aptamer AFB1 After being activated by mixing with TECP in equal volumes, the mixture was added to the Au-Ag Janus NPs solution obtained in step S2.2 and mixed evenly. The mixture was incubated at room temperature. After the incubation was completed, the unconnected aptamers were removed by centrifugation and resuspended in PBS buffer to obtain the aptamer-functionalized signal probe Au-Ag Janus NPs-apt AFB1 Solution; S3. Preparation of SERS aptasensor The capture probe obtained in step S1 and the signal probe obtained in step S2 are mixed in equal volumes, incubated statically, centrifuged and then resuspended to obtain the SERS aptamer sensor SiO2@AgNPs-Au-Ag Janus NPs.

3. The method for preparing the label-free functionalized SERS aptamer sensor according to claim 2, characterized in that: In step S1.1, The volume ratio of ethanol to NH3·H2O is 8 mL:3 mL; the volume ratio of ethanol to tetraethyl orthosilicate TEOS is 8 mL:2 mL; the stirring time is 24 hours; and the centrifugal speed is 5000 rpm.

4. The method for preparing the label-free functionalized SERS aptamer sensor according to claim 2, characterized in that: In step S1.2, The concentration of NH3·H2O is 5wt%; the volume of AgNO3 is 4mL, the concentration is 0.4M; the total volume diluted with water is 12mL; the ratio of SiO2 to ethanol is 0.25g:10mL; the [Ag(NH3)2] + The volume is 10 mL; the stirring time is 1 hour; and the centrifugal speed is 4000 rpm.

5. The method for preparing the label-free functionalized SERS aptamer sensor according to claim 2, characterized in that: In step S1.3, The incubation time is 12 hours; The pH value of the PBS buffer is 7.2-7.4; The concentration of the activated aptamer complementary chain is 100 μM; The mixed volume of the aptamer complementary chain and SiO2@AgNPs was 10 μL; The aptamer complementary chain nucleic acid sequence is 5'-SH-CAGAGAGACAACACGTGCCCAAC-3', and a thiol group is modified at the 5' end.

6. The method for preparing the label-free functionalized SERS aptamer sensor according to claim 2, characterized in that: In step S2.1, the concentration of HAuCl4·4H2O is 1wt%, and the volume ratio of HAuCl4·4H2O to deionized water is 0.5mL:49.5mL; the concentration of trisodium citrate solution is 1wt%, and the volume is 1mL; In step S2.2, the volume ratio of the AuNPs solution to deionized water is 1 mL:9 mL; the volume of the 2-mercapto-5-benzimidazole carboxylic acid MBIA solution is 200 μL, and the solution is maintained in a 60°C water bath environment for 2 hours; the volume of the hydroquinone HQ solution is 200 μL; the volume of the AgNO3 solution is 300-1800 μL, preferably, the volume of the AgNO3 solution is 600 μL, and the centrifugal speed is 6000 rpm; the re-dissolved volume of the solution after centrifugation is 2 mL; In step S2.3, the incubation time is 12 hours; The concentration of the activated aptamer is 100 μM; The mixed volume of the aptamer and Au-Ag Janus NPs was 5 μL; The aptamer Apt AFB1 The nucleotide sequence is: 5'-SH-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3', with thiol modification at the 5' end.

7. The method for preparing the label-free functionalized SERS aptamer sensor according to claim 2, characterized in that: In step S3, The volume ratio of the capture probe to the signal probe is 1:1 to 1:5, preferably, the volume ratio of the capture probe to the signal probe is 1:4; the incubation time is 0 to 3 hours, preferably, the incubation time is 2 hours.

8. Use of a SERS aptamer sensor prepared according to the label-free functionalized SERS aptamer sensor of claim 1 or the preparation method of the label-free functionalized SERS aptamer sensor of any one of claims 2 to 7 in detecting aflatoxin B1.

9. A method for detecting aflatoxin B1 using a label-free functionalized SERS aptamer sensor, characterized in that: The label-free functionalized SERS aptamer sensor comprises the label-free functionalized SERS aptamer sensor according to claim 1, or the label-free functionalized SERS aptamer sensor prepared by the preparation method of the label-free functionalized SERS aptamer sensor according to any one of claims 2 to 7, and the detection method comprises the following steps: (1) The label-free functionalized SERS aptamer sensor was mixed with a standard solution of aflatoxin B1, and after a capture reaction, the Raman spectrum was collected; (2) Establishing a standard curve based on the relationship between the concentration of aflatoxin B1 standard solution and the characteristic Raman peak of the label-free functionalized SERS aptamer sensor; (3) Detecting the content of aflatoxin B1 in the sample to be tested: taking a sample solution of the sample to be tested and mixing it with the label-free functionalized SERS aptamer sensor for detecting aflatoxin B1, collecting Raman spectra according to the method of step (1), and calculating the content of aflatoxin B1 in the sample to be tested according to the standard curve of step (2).

10. The method for detecting aflatoxin B1 using a label-free functionalized SERS aptamer sensor according to claim 9, characterized in that: The concentration of the aflatoxin B1 standard solution is 0.0001-100 ng / mL; the standard curve is y=-3286.44x+11927.26, R 2 =0.990, and the detection limit of AFB1 is 0.00134 μg / kg.

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