A rigid sers aptamer sensor and a preparation method and application thereof

Abstract

The application discloses a rigid SERS aptamer sensor and a preparation method and application thereof, and belongs to the technical field of rapid evaluation of food safety. The rigid SERS enhancement substrate is prepared by first depositing a gold film on conductive glass and then dropping graphene oxide; the specific signal probes of two target detection objects are prepared by respectively adsorbing the Raman signal molecules 4-mercaptobenzonitrile and 4-mercaptopyridine with distinguishability in characteristic peaks in the gold core-silver shell shell structure and further respectively modifying the zearalenone aptamer and the ochratoxin aptamer; the SERS spectra of the SERS aptamer sensor detecting zearalenone and ochratoxin at an excitation wavelength are collected, a standard curve of the spectral intensity at the characteristic Raman peak and the content of zearalenone and ochratoxin is established, and the simultaneous rapid quantitative detection of zearalenone and ochratoxin in grains or food is realized.

Description

Technical Field

[0001] This invention relates to a rigid SERS aptamer sensor, its preparation method and application, and more particularly to a rigid SERS aptamer sensor for simultaneous detection of zearalenone and ochratoxin, its preparation method and application, belonging to the field of rapid food safety evaluation technology. Background Technology

[0002] For single mycotoxins, various surface-enhanced Raman spectroscopy (SERS) methods have been developed, exhibiting a good linear relationship between Raman signal intensity and analyte concentration, and demonstrating high sensitivity. However, existing SERS biosensor detection systems are commonly liquid and typically detect only one type of mycotoxin. Long-term storage can alter their stability and homogeneity, affecting detection accuracy and failing to meet the needs of detecting samples contaminated with multiple mycotoxins in practical applications. Current conventional techniques for mycotoxin detection in food mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). These methods show good accuracy and specificity, but their sensitivity is limited. Therefore, developing a stable SERS biosensor with specific recognition capabilities capable of simultaneously detecting several mycotoxins is of great significance and promising development potential for improving detection efficiency and expanding the application areas of SERS spectroscopy.

[0003] Surface-enhanced Raman spectroscopy (SERS) utilizes the localized electromagnetic field formed by reinforcing a substrate with precious metals such as gold and silver. This enhances the Raman signal intensity of the analyte, enabling detection at the single-molecule level. Traditional SERS-enhancing substrates are typically prepared through chemical synthesis, i.e., dispersing metal colloids in an aqueous phase. This method is limited by the large consumption of chemicals, complex and lengthy preparation processes, and poor hotspot reproducibility due to operational randomness. Currently, the selection and modification of SERS active groups for different toxins remains challenging, and the complexity of the sample matrix can also interfere with simultaneous detection. Therefore, a tool or method for the simultaneous detection of two toxins is urgently needed in this field. Summary of the Invention

[0004] To address some shortcomings in existing technologies, this invention provides a rigid SERS aptamer sensor for the simultaneous detection of zearalenone and ochratoxin, along with its preparation method and applications. This invention utilizes a rigid SERS-enhanced substrate and combines two specific signal probes to construct a detection method for the simultaneous detection of these two toxins, successfully achieving the simultaneous detection of zearalenone and ochratoxin in corn. The SERS aptamer sensor described in this invention features a simple preparation process, high sensitivity, and is suitable for the rapid and simultaneous detection of zearalenone and ochratoxin in grains and their products.

[0005] This invention involves depositing a layer of gold nanoparticles on the surface of conductive glass ITO using electrodeposition. Graphene oxide (GO) is then coated onto this layer as a rigid SERS-enhancing substrate. Raman signal molecules 4-mercaptobenzonitrile (TBN) and 4-mercaptopyridine (4-MPy), with distinguishable characteristic peaks, are adsorbed into the gold core-silver shell structure. These are further modified with zearalenone and ochratoxin aptamers, respectively, to serve as specific signal probes for the two target analytes. The aptamers of the signal probes adhere to the rigid SERS substrate surface via non-covalent interactions (π-π bonds) with the graphene oxide on the ITO / AuNPs / GO surface, generating a SERS-enhancing effect. The preferential and specific binding between the target analyte and the aptamer causes the signal probes to dissociate from the rigid substrate surface. By measuring the Raman response intensity of the SERS probes, a quantitative model is established, enabling rapid quantitative prediction of the contents of zearalenone and ochratoxin.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a rigid SERS aptamer sensor, which adsorbs signal molecules 4Mpy and TBN and can simultaneously detect zearalenone and ochratoxin.

[0008] This invention provides a method for fabricating the rigid SERS aptamer sensor, comprising the following steps:

[0009] Fabrication of rigid SERS-reinforced substrates ITO / AuNPs / GO:

[0010] Conductive glass ITO was ultrasonically dried in acetone, ethanol and ultrapure water respectively. Gold nanoparticles AuNPs were deposited on the ITO surface using HAuCl4 aqueous solution as electrolyte to prepare ITO / AuNPs. Then, graphene oxide solution was dropped onto the surface of the ITO / AuNPs and dried to obtain a rigid SERS-reinforced substrate ITO / AuNPs / GO.

[0011] The concentration of the HAuCl4 solution is 10 mM; the concentration of the graphene oxide solution is 0.00625-0.25 mg / mL, and the volume is 200 μL; preferably, the concentration of the graphene oxide solution is 0.16 mg / mL.

[0012] Furthermore, the ultrasonic time is 10 minutes, the drying condition is drying at 40°C, the electrodeposition condition is electrodeposition at a potential of -1.5V for 200 seconds, the conductive glass (ITO) is 1×2 cm in size, and the immersion area during AuNP deposition is 1×1 cm, and the drying is performed at 40°C.

[0013] Preparation of specific SERS probes Au-4MPy@AgNPs-Apt(OTA) and Au-TBN@AgNPs-Apt(ZEN):

[0014] (1) Preparation of gold nanoparticle solution:

[0015] Trisodium citrate was rapidly added to a boiling HAuCl4 aqueous solution, and the solution was kept boiling until the color turned wine red and remained so for a certain period of time. The solution was then cooled to room temperature and concentrated by centrifugation to obtain a gold nanoparticle solution.

[0016] The volume ratio of the HAuCl4 aqueous solution to the trisodium citrate aqueous solution is 4:3; the concentration of the HAuCl4 aqueous solution is 0.25 mM, and the concentration of the trisodium citrate aqueous solution is 1 wt%; the solution is boiled until the color turns wine red and remains so for 10 min; the volume of the concentrated gold nanoparticle solution is 10 mL.

[0017] Furthermore, according to an embodiment of the present invention, the amounts of the HAuCl4 aqueous solution and the trisodium citrate aqueous solution are 20 mL and 15 mL, respectively.

[0018] (2) Preparation of Au-4MPy and Au-TBN adsorbed with signal molecules 4MPy and TBN:

[0019] In the same volume of gold nanoparticle solution prepared in step (1) of S2, 4 MPy of alcoholic solution of TBN is added respectively. After adsorbing signal molecules under slight vibration, the unadsorbed Raman signal molecules are removed by centrifugation, and the mixture is redispersed (preferably by adding pure water for mixing and dispersion).

[0020] The final concentrations of the two signaling molecules, 4MPy and TBN, in the mixed solution were both 10 μM; the adsorption time for the signaling molecules was 2 h; and the centrifugation conditions were 10,000 rpm for 10 min.

[0021] (3) Synthesis of silver-coated Raman signal probes Au-4MPy@AgNPs and Au-TBN@AgNPs:

[0022] In step (2), trisodium citrate solution, AgNO3 solution, and ascorbic acid solution were added to the solutions Au-4MPy and Au-TBN that were adsorbed with Raman signal molecules, respectively, and the mixture was stirred vigorously for 10 min to obtain Raman signal probes Au-4MPy@AgNPs and Au-TBN@AgNPs.

[0023] The volume ratio of the solution adsorbed with Raman signal molecules, trisodium citrate solution, AgNO3 solution, and ascorbic acid solution is 5:0.8:0.8-1.4:2; wherein the concentration of the trisodium citrate solution is 8.8 mM, the concentration of the AgNO3 solution is 38.8 mM, and the concentration of the ascorbic acid solution is 10 mM.

[0024] Preferably, the volume ratio of the solution adsorbed with Raman signal molecules, trisodium citrate solution, AgNO3 solution, and ascorbic acid solution is 5:0.8:1.1:2.

[0025] Furthermore, according to embodiments of the present invention, the volume of the solution adsorbed with different Raman signal molecules is 5 mL, the volume of the trisodium citrate solution is 800 μL, the volume of the AgNO3 solution is set to 800-1400 μL, and the volume of the ascorbic acid solution is 2 mL; preferably, the volume of the AgNO3 solution is 1100 μL.

[0026] (4) Preparation of aptamer-functionalized signal probes Au-4MPy@AgNPs-Apt(OTA) and Au-TBN@AgNPs-Apt(ZEN):

[0027] The thiol-modified aptamers were activated using TCEP solution. An equal volume of TCEP aqueous solution was added to the aptamer solution and reacted for 1 h to reduce the disulfide bond of the thiol to a monothiol. The activated aptamers were then added to the corresponding Raman signal probe solutions and the mixture was incubated at room temperature for 12 h. After incubation, the mixture was centrifuged at low speed for 10 min to remove unconnected aptamers and resuspended in Tris-HCl buffer and stored at 4 °C.

[0028] The molar ratio of the aptamer solution to the TCEP solution is 1:100; the concentration of the aptamer solution is 10 μM and the volume is 100 μL; the volume of the Raman signal probe solution is 1 mL; the Tris-HCl buffer solution is a mixture of Tris-HCl, NaCl, and KCl, wherein the concentrations of each component are 25 mM Tris-HCl, 140 mM NaCl, and 5 mM KCl, and the pH is 7.4.

[0029] The aptamers include ZEN-Apt, an aptamer against zearalenone (ZEN), and OTA-Apt, an aptamer against ochratoxin (OTA); the sequence of ZEN-Apt is as follows:

[0030] SH-5'-AGCAGCACAGAGGTCAGATGTCATTCTATGGTACATTACTATCTCTGTAATGTGATATGCCTATGCGTGCTACCGTGAA-3', with a thiol group modified at the 5' end.

[0031] The OTA-Apt sequence is as follows:

[0032] SH-5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3', with a thiol group modified at the 5' end.

[0033] Fabrication of rigid SERS aptamer sensors:

[0034] Two SERS probes, Au-4MPy@AgNPs-Apt (OTA) and Au-TBN@AgNPs-Apt (ZEN), were mixed and bonded to an ITO / AuNPs / GO surface to form a rigid SERS aptamer sensor assembly.

[0035] The volume ratio of Au-4MPy@AgNPs-Apt(OTA) to Au-TBN@AgNPs-Apt(ZEN) is 3.5-6.5:6.5-3.5, preferably 6:4.

[0036] The present invention also provides a method for simultaneously detecting zearalenone and ochratoxin, the method being based on the aforementioned rigid SERS aptamer sensor, and the method comprising the following steps:

[0037] S1. The prepared rigid SERS aptamer sensor is immersed in a mixed solution of standard OTA and standard ZEN. After the capture reaction, Raman spectra are collected.

[0038] The concentration of the standard OTA is 0.001-20 ng / mL, the concentration of the standard ZEN is 0.1-100 ng / mL, the mixed solution is prepared by combining two standards with concentrations from low to high in pairs, and the volume ratio of the two standards is 1:1; the capture time is 1 h.

[0039] S2. Establish a standard curve based on the relationship between the mixed solution of standard OTA and ZEN and the characteristic Raman peaks of their corresponding signal molecules 4Mpy and TBN.

[0040] S3. Detect the content of zearalenone and ochratoxin in the sample to be tested: Mix the sample to be tested with a rigid SERS aptamer sensor, collect Raman spectra according to the method in step S1, and calculate the concentration of zearalenone and ochratoxin in the sample to be tested according to the standard curve in step S2.

[0041] The content of zearalenone and ochratoxin in the sample can also be further calculated based on the measured concentration. Taking zearalenone as an example, the content is calculated in the following way in this invention:

[0042] The zearalenone content in actual positive maize samples is calculated according to formula (1):

[0043]

[0044] X: The content of zearalenone in the sample, in micrograms per kilogram (μg / kg).

[0045] ρ: Concentration of zearalenone in the sample solution, expressed in nanograms per milliliter (ng / mL).

[0046] V: The volume of the sample solution added, in milliliters (mL).

[0047] M: Sample weight, in grams (g).

[0048] The present invention also provides the application of the rigid SERS aptamer sensor or the detection method in the simultaneous detection of zearalenone and ochratoxin in food or grains.

[0049] The beneficial effects of this invention are:

[0050] (1) The rigid conductive glass (ITO / AuNPs) with gold nanoparticles deposited in this invention requires simple raw materials and has a simple preparation process. Rigid conductive glass (ITO / AuNPs) has the advantages of small size and light weight, making it easy to carry and use. It can be used as a key component of portable detection devices. In addition, by depositing gold nanoparticles onto conductive glass through electrodeposition, the synergistic effect of gold nanoparticles and conductive glass ITO is fully utilized. This not only improves the sensitivity of the substrate and reduces the interference of background noise, but also achieves better controllability and repeatability, effectively improving the performance and reliability of toxin detection.

[0051] (2) This invention relates to coated graphene oxide (GO), which possesses outstanding electronic properties and thermal conductivity, a large specific surface area, and strong biocompatibility. In this invention, graphene oxide can effectively enhance the Raman signal intensity of analytes, reduce background noise, and suppress background fluorescence. In particular, the combination of gold nanoparticles and graphene makes the signal enhancement effect more significant, effectively suppressing background fluorescence interference and further improving the sensitivity and accuracy of detection. In addition, graphene has good adsorption properties and can adsorb a variety of organic molecules, allowing the Apt on the signal probe to be well connected to the rigid substrate, improving the stability of detection.

[0052] (3) The rigid SERS aptamer sensor and detection method of this invention can achieve simultaneous and rapid detection of zearalenone and ochratoxin. Simply mix the rigid substrate with the two probes connected to it with the sample, collect the signal, and establish a correlation between the Raman characteristic peaks of the signal molecules and the concentration of the corresponding target analytes to quickly complete the simultaneous detection and obtain accurate results. The detection process can be completed within 3 minutes, with a detection limit of 0.476 μg / kg for OTA and 0.374 μg / kg for ZEN. For actual positive samples, the method of this invention shows no significant difference from the traditional HPLC method (P>0.05), exhibiting good stability with an RSD (n=7) of 1.7 to 5.55. By simply preparing and detecting the peak intensity, the quality and safety of grains can be determined, achieving simultaneous, rapid, and intelligent detection of zearalenone and ochratoxin. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of a simultaneous detection technique for SERS aptamers of zearalenone and ochratoxin.

[0054] Figure 2 Characterization of the ITO / AuNPs / GO preparation process; where (a) is a TEM image of ITO / AuNPs, (b) is a TEM image of ITO / AuNPs / GO, (c) is a Raman spectrum of the ITO / AuNPs / GO preparation process, and (d) is an atomic force microscopy image of ITO / AuNPs, (e) ITO / AuNPs / GO, and (f) ITO / AuNPs / GO as adsorption signal probes on rigid reinforced substrates.

[0055] Figure 3 shows the characterization of the fabrication process of two signal probes with gold and silver core-shell structures; where (a) is the TEM image of AuNPs at ×10 magnification, (b) is the TEM image of AuNPs at ×50 magnification, (c) is the particle size distribution of AuNPs, (d) is the TEM image of Au@AgNPs at ×10 magnification, (e) is the TEM image of Au@AgNPs at ×50 magnification, (f) is the particle size distribution of Au@AgNPs, (g) is the UV-Vis image of the fabrication process of the two signal probes, (h) is the Raman enhancement characterization image of the two signal probes, and (i) is the potential change diagram during the fabrication process of the OTA signal probe as an example.

[0056] Figure 4 shows the construction of a detection system for simultaneous detection of OTA and ZEN using two Raman signal probes on a rigid substrate. Among them, (a) is the Raman characterization diagram of the SERS aptamer sensing component during construction, (b) is the SEM image of the SERS aptamer sensing component, (c) is the elemental analysis diagram of the SERS aptamer sensing component, (d) is the EIS variation diagram of the SERS aptamer sensing component during construction, (e) is the CV curve diagram of the SERS aptamer sensing component during construction and detection, and (f) is the UV-Vis characterization diagram of the optimized modification process of the two signal probe aptamers.

[0057] Figure 5 shows the optimization of the detection system for OTA and ZEN using a rigid substrate combined with a Raman signal probe. Among them, (a) is the optimized response surface diagram of the OTA signal probe SERS aptamer sensor, (b) is the optimized response surface diagram of the ZEN signal probe SERS aptamer sensor, (c) is the Raman spectrum of OTA standard detected by the OTA signal probe SERS aptamer sensor, (d) is the Raman spectrum of ZEN standard detected by the ZEN signal probe SERS aptamer sensor, (e) is the standard curve of the OTA signal probe SERS aptamer sensor, and (f) is the standard curve of the ZEN signal probe SERS aptamer sensor.

[0058] Figure 6 The results of optimizing the detection system for simultaneous detection of OTA and ZEN using a dual-specific Raman signal probe.

[0059] Figure 7 shows the detection results of OTA and ZEN simultaneously detected by a rigid substrate combined with two Raman signal probes; where (a) is the Raman spectrum of the mixed standard of OTA and ZEN detected by the SERS aptamer sensor of the two signal probes, (b) is the standard curve of OTA by the SERS aptamer sensor of the two signal probes, (c) is the standard curve of ZEN by the SERS aptamer sensor of the two signal probes, (d) is the detection result of a single spiked sample by the SERS aptamer sensor of the two signal probes, (e) is the detection result of a mixed spiked sample by the SERS aptamer sensor of the two signal probes, and (f) is the specificity detection of the SERS aptamer sensor of the two signal probes. Detailed Implementation

[0060] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention will be described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0061] In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. The processes involved in the embodiments that are not described in detail are those that can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0062] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0063] Example 1: Fabrication of a rigid substrate-bonded signal probe SERS aptamer sensor

[0064] (1) Preparation of rigid substrate ITO / AuNPs / GO

[0065] Conductive glass (ITO, 1×2 cm) was ultrasonicated for 10 min in acetone, ethanol, and ultrapure water, respectively, and then dried at 40 °C. AuNPs were deposited on the exposed ITO glass (immersion area 1×1 cm) surface using a potentiostatic electrodeposition method with HAuCl4 aqueous solution as the electrolyte at a concentration of 10 mM and a potential of -1.5 V for 200 s. After electrodeposition, the surface was immersed in ultrapure water for 30 s twice to remove surface residues, thus obtaining conductive glass ITO / AuNPs with deposited gold nanoparticles.

[0066] Then, 200 μL of graphene oxide (GO) solution with a concentration of 0.16 mg / mL was dropped onto the ITO / AuNPs surface and dried at 40 °C to obtain a rigid SERS-reinforced substrate ITO / AuNPs / GO.

[0067] TEM images of rigid substrates ITO / AuNPs / GO prepared using the above method show that ITO / AuNPs ( Figure 2 a) The surface is relatively rough, and obvious granular texture can be observed. After being covered with graphene, ITO / AuNPs / GO ( Figure 2 b) The surface particle outline is blurred. This is because GO, as an ultrathin film, has high transmittance. The flexible GO film with folds and wrinkles is attached around the gold nanoparticles, which blurs the edges of AuNPs. In particular, the hydrophilicity of the AuNPs film promotes the rapid and uniform diffusion of the GO solution.

[0068] SERS spectra were collected during the ITO substrate modification process. Figure 2 c shows that there is almost no interference signal on bare ITO and ITO / AuNPs, and the spectrum of ITO / AuNPs / GO shows that GO is located at 1332 cm⁻¹. -1 (D belt) and 1594cm -1 Two distinct Raman characteristic peaks at (G band).

[0069] The preparation process of the rigid substrate ITO / AuNPs / GO was characterized by atomic force microscopy. Figure 2 The image shows a microscopic image of ITO / AuNPs, revealing clear contours on the gold film surface. Image processing showed an average thickness of 15.39 nm. Further GO coating on the surface resulted in a more detailed microscopic image. Figure 2 As shown in Figure e, a large area of ​​smooth, wrinkled layer can be observed covering the surface. The morphological changes indicate that GO has successfully adhered to the ITO / AuNPs surface. Furthermore, due to the GO coating, the thickness increases to 19.43 nm. When the signal probe is further attached, obvious granular protrusions appear on the smooth layer surface, and the thickness increases to 25.63 nm. Figure 2 f).

[0070] (2) Preparation of signal probes Au-4MPy@AgNPs-Apt(OTA) and Au-TBN@AgNPs-Apt(ZEN)

[0071] Preparation of gold nanoparticle solution: Under vigorous stirring, 15 mL of 1 wt% trisodium citrate solution was rapidly added to 20 mL of 0.25 mM HAuCl4 aqueous solution heated to boiling. The solution was kept boiling until the color turned wine red and continued for 10 min. After cooling to room temperature, the solution was concentrated to 10 mL by centrifugation to obtain gold nanoparticle solution.

[0072] The process of linking gold nanoparticles to signal molecules involves preparing Au-4MPy and Au-TBN, which are adsorbed with signal molecules 4MPy and TBN. Two centrifuge tubes are each filled with the same volume of gold nanoparticle solution, and alcohol solutions of 4MPy and TBN are added to each tube to achieve a concentration of 10 μM. After adsorbing the signal molecules for 2 hours under gentle vibration, the tubes are centrifuged at 10,000 rpm for 10 minutes to remove unadsorbed Raman signal molecules. The mixture is then redispersed in pure water to the original volume (the total volume of the gold nanoparticle solution and the alcohol solution of the signal molecules before centrifugation).

[0073] The silver-shell encapsulation process: This involves the synthesis of Au-4MPy@AgNPs and Au-TBN@AgNPs coated with silver shells. 5 mL of solutions of Au-4MPy and Au-TBN, each adsorbed with Raman signal molecules 4Mpy and TBN, were added to 800 μL of 8.8 mM trisodium citrate solution, followed by 1100 μL of 38.8 mM AgNO3 solution. Finally, 2 mL of 10 mM ascorbic acid solution was added, and the mixture was stirred vigorously for 10 min to obtain the Raman signal probes Au-4MPy@AgNPs and Au-TBN@AgNPs.

[0074] Aptamer-functionalized Au-4MPy@AgNPs and Au-TBN@AgNPs: The thiol-modified aptamers were activated using TCEP solution. An equal volume of TCEP aqueous solution (molar ratio of aptamer solution to TCEP solution was 1:100) was added to the aptamer solution and reacted for 1 h to reduce the disulfide bond of the thiol to a monothiol. Then, 100 μL of the activated aptamer at a concentration of 10 μM was added to 1 mL of the corresponding Raman signal probe solution and the mixture was incubated at room temperature for 12 h. After incubation, the mixture was centrifuged at low speed for 10 min to remove unlinked aptamers and resuspended in Tris-HCl buffer (25 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, pH 7.4) to 1 mL to obtain the aptamer-functionalized signal probes Au-4MPy@AgNPs-Apt (OTA) and Au-TBN@AgNPs-Apt (ZEN).

[0075] The aptamers include ZEN-Apt, an aptamer against zearalenone (ZEN), and OTA-Apt, an aptamer against ochratoxin (OTA); the sequence of ZEN-Apt is as follows:

[0076] SH-5'-AGCAGCACAGAGGTCAGATGTCATTCTATGGTACATTACTATCTCTGTAATGTGATATGCCTATGCGTGCTACCGTGAA-3', with a thiol group modified at the 5' end.

[0077] The OTA-Apt sequence is as follows:

[0078] SH-5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3', with a thiol group modified at the 5' end.

[0079] In this embodiment, the morphology and size of the nanoparticles were characterized using transmission electron microscopy and a laser particle size analyzer. Figure 3a -f), the results show that the synthesized gold nanoparticle solution AuNPs has good dispersibility ( Figure 3a , 3b The average diameter is 18.63 nm. Figure 3c Since gold nanoparticles (AuNPs) are prepared by reduction with trisodium citrate, their surface contains a large number of citrate anions, thus carrying a negative charge. Figure 3i After the adsorption of signal molecules and the reduction growth of silver shells by silver nitrate were completed on the basis of AuNPs, the prepared Au-4MPy@AgNPs and Au-TBN@AgNPs nanoparticles showed a certain degree of aggregation, and the silver shell structure could be observed on the outer layer of AuNPs. Figure 3d ,3e The average diameter increased to 25.13 nm. Figure 3f And the potential rose to -32.13mV. Figure 3i ).

[0080] The characterization results of the UV-Vis signal probe synthesis process are as follows: Figure 3g The synthesized AuNPs exhibited a maximum absorption peak at 535 nm. After further adsorption of Raman signal molecules and growth of a silver shell, the absorption peak of the AuNPs at 535 nm blue-shifted to 505 nm. Additionally, an absorption peak at 380 nm appeared for AgNPs, indicating that the silver shell significantly affected the dielectric properties of the AuNPs. Furthermore, the SERS enhancement effect of the core-shell structure was illustrated by acquiring SERS spectra. Figure 3h As shown, AuNPs themselves do not have obvious Raman characteristic peaks, but when AuNPs adsorb signal molecules, they exhibit a certain Raman enhancement effect. When a silver shell is further grown, the enhancement effect on signal molecules is more significant than that of direct adsorption on the surface of AuNPs. Therefore, embedding signal molecules into Au@AgNPs to form a bimetallic core-shell nanostructure can exert an effective Raman synergistic enhancement effect and can be used as a signal probe.

[0081] The target analyte was identified using an aptamer-functionalized signal probe, and the aptamer functionalization process was analyzed using UV-Vis spectroscopy and Zeta potential measurements. Figure 3g As shown, both the OTA and ZEN aptamers exhibit a characteristic UV absorption peak at 256 nm. After functionalization of Au-TBN@AgNPs and Au-4MPy@AgNPs, in addition to their inherent gold-silver core-shell characteristic absorption peaks, characteristic absorption peaks of the aptamers also appeared at 256 nm. Taking the OTA aptamer modification process as an example, the potential change was measured. Due to the negatively charged phosphate group on the aptamer, the potential of the signal probe decreased to -38.01 mV. Figure 3i This indicates that the aptamer modification was successful.

[0082] The above experiments demonstrate that the addition of gold nanoparticles improves detection sensitivity and reduces background signal interference. When gold nanoparticles are deposited on conductive glass, the plasmon resonance effect on the surface of the gold nanoparticles enhances the signal of the target molecules, making the detection signal more prominent relative to background noise, thereby reducing background noise interference. This deposition forms a dense and regular capping layer, which can suppress the generation and propagation of background signals, further reducing background noise interference.

[0083] (3) Fabrication of rigid SERS aptamer sensors

[0084] In the construction of a system for the simultaneous detection of ochratoxin and zearalenone on a rigid SERS-enhanced substrate, the detection mechanism involved the non-covalent interaction between the ochratoxin aptamer-modified signal probe Au-4MPy@AgNPs-Apt(OTA) and the zearalenone aptamer-modified signal probe Au-TBN@AgNPs-Apt(ZEN) and graphene, respectively, to create ITO / AuNPs / GO-Apt(OTA)-AgNPs@4MPy-Au and ITO / AuNPs / GO-Apt(ZEN)-AgNPs@TBN-Au aptamer sensing groups. This composite substrate significantly improved the Raman response intensity of the Raman signal molecules, with 4MPy and 1005cm⁻¹ being selected as the optimal values. -1 2227cm -1 The intensity of the characteristic peak at a given location is used as the intensity of the response signal. In the absence of the target object OTA and ZEN, the characteristic peak of the aptamer sensing component exhibits a strong Raman response. However, when the target object is present, the high affinity of the aptamer for the target object causes the signal probe to dissociate, resulting in a decrease in the intensity of the corresponding characteristic peak.

[0085] Raman characterization of the SERS aptamer sensor construction process is shown in Figure 4a. As shown in Figure 4a(1), the Raman signal molecules 4MPy and TBN are at 1100 cm⁻¹. -1 1200cm -1 and 1600cm -1 Raman characteristic peaks are present in the vicinity, but the peak positions overlap or the peak shapes are bifurcated, while those located at 1005 cm⁻¹ are... -1 The location belongs to 4 MPy and is located at 2227 cm. -1 The characteristic peaks belonging to TBN are single peaks with no overlap or interference. Therefore, these two characteristic peaks were selected as the intensity analysis peaks of the two signal probes, respectively. Figure 4a (2) and Figure 4a (3) SERS spectra of 4MPy and OTA signal probes (Au-4MPy@AgNPs-Apt(OTA)) and TBN and ZEN signal probes (Au-TBN@AgNPs-Apt(ZEN)) solutions directly added to ITO / AuNPs / GO were obtained. It was found that using the signal probes showed a stronger SERS enhancement effect compared to directly adding the signal molecule solution, indicating that the introduction of the signal probes greatly improved the SERS signal enhancement. Through recombination with the rigid ITO / AuNPs / GO substrate, the signal probes successfully reduced the interference of the background signal.

[0086] Two signal probes (Au-4MPy@AgNPs-Apt(OTA) and Au-TBN@AgNPs-Apt(ZEN)) were attached to SEM images of ITO / AuNPs / GO. Figure 4b Dispersed signal probes can be clearly observed on the ITO / AuNPs / GO surface. Elemental analysis of the SEMP-captured area also shows... Figure 4c For the rigid substrate surface with attached signal probes, in addition to the carbon, oxygen, and gold elements that belong to graphene and electrodeposition, there are also silver elements that belong to the silver shell structure of the signal probes, indicating that the signal probes have been successfully attached.

[0087] Furthermore, the substrate assembly process was verified using EIS and CV analyses, and the EIS value showed a positive correlation with the magnitude of the capacitive arc on the curve. Figure 4d In the figure, compared with the arc size of ITO, the ITO / AuNPs ratio is significantly reduced, indicating a substantial decrease in impedance. CV curve ( Figure 4e The EIS and CV curves reflect the current changes on the rigid substrate surface. ITO / AuNPs exhibit a higher redox peak than ITO, reflecting changes in electron transfer during modification and indicating an expansion of the active region on the substrate surface. Modification with the insulator GO hinders electron transport, leading to a decrease in the redox peak. After the signal probe self-assembles onto the ITO / AuNPs / GO surface via covalent π-π bonds, the negatively charged phosphate backbone of the aptamer restricts electron transfer on the substrate surface, further reducing the redox peak. When the sensor was immersed in the target solution (OTA = 50 ng / mL, ZEN = 100 ng / mL) for a period of time, a re-measurement of its CV curve revealed that the ionization of the signal probe slightly increased the redox peak compared to before immersion. The EIS and CV curve results demonstrate the successful construction of the ITO / AuNPs / GO rigid substrate and the ability of the signal probe to be loaded and ionized on its surface to construct the aptamer sensor.

[0088] (4) Optimization of a rigid substrate combined with a single signal probe detection system

[0089] To achieve simultaneous detection of two mycotoxins using an aptamer sensor, the optimal parameters for a single mycotoxin detection system using an aptamer sensor were first explored:

[0090] The optimization results of the two signal probe aptamer modification processes are presented in Figure 4f The left side of the figure shows the results of concentration optimization for OTA and ZEN aptamers, respectively. To demonstrate the construction of signal probes through incubation of aptamers with core-shell structures, pure aptamer solutions before incubation, incubation solutions with aptamer saturation (different concentrations of aptamer solution were added to Au-4MPy@AgNPs and Au-TBN@AgNPs solutions, and their absorbance values ​​were measured; they first increased and then decreased, with the inflection point corresponding to the saturation concentration), and a control solution with the same volume of aqueous solution were incubated and then subjected to UV-Vis analysis. The results are shown in [Figure number missing]. Figure 4fOn the right, the absorbance value decreased significantly after incubation, while the absorbance value of the control blank group decreased slightly compared with the original solution due to the concentration decrease caused by dilution. The results show that the saturation incubation concentration of OTA aptamer is 800 nM, and the saturation concentration of ZEN aptamer incubating Au-TBN@AgNPs is 700 nM.

[0091] Based on the aptamer optimization results, the experimental parameters for constructing aptamer sensors using single signal probes were optimized through response surface methodology. The three optimization factors were the concentration of graphene added to the surface of ITO / AuNPs (0.00625-0.25 mg / mL), the amount of AgNO3 added during the preparation of the signal probe that affects the thickness of the silver shell (800 μL-1400 μL), and the immersion time of ITO / AuNPs / GO in the probe solution during assembly with the signal probe (0.5-2 h). The intensity of the Raman characteristic peak of the sensor was used as the indicator of optimization.

[0092] Based on response surface methodology and experimental results, the amount of AgNO3 added did not significantly affect the signal probe's adhesion to the rigid substrate to produce a good Raman response. Therefore, the amount of AgNO3 added was set as an intermediate value of 1100 μL.

[0093] The response surface results of the Au-4MPy@AgNPs aptamer sensor (a single-signal rigid SERS aptamer sensor obtained by combining Au-4MPy@AgNPs-Apt(OTA) with a rigid substrate ITO / AuNPs / GO) are as follows: Figure 5a As shown, the intensity of the characteristic peak corresponding to 4 MPy increases with increasing graphene concentration up to 0.15 mg / mL and immersion time up to 1 h, and then begins to decrease. Excessive graphene layer is not conducive to the formation of hotspot space between the rigid substrate and the signal probe, and a long immersion time can also lead to the ionization of the signal probe. According to the matrix results of the function, the optimal parameter combination is a graphene concentration of 0.1592 mg / mL, an immersion time of 1.37 h, and an AgNO3 addition of 1100 μL. Figure 5b The results show the response surface methodology of the Au-TBN@AgNPs aptamer sensor (a single-signal rigid SERS aptamer sensor obtained by combining Au-TBN@AgNPs-Apt(ZEN) with a rigid substrate ITO / AuNPs / GO) as detected by ZEN. The optimal parameter combination can be observed to be a graphene concentration of 0.1692 mg / mL, a soaking time of 1.43 h, and an AgNO3 addition of 1100 μL.

[0094] The graphene oxide used in this invention has a low fluorescence background, effectively absorbing surrounding background fluorescence and reducing background noise interference. Furthermore, the high specific surface area and adsorption properties of graphene oxide enable it to adsorb and remove impurities from the sample, further reducing background noise interference. The adsorption properties of the graphene surface allow the Apt on the signal probe to be firmly bonded to the rigid substrate, providing a stable detection platform. This helps prevent probe displacement or detachment, ensuring signal stability and repeatability.

[0095] (5) Establishment of standard curve for single signal probe SERS aptamer sensor

[0096] Under optimal experimental parameter combinations, OTA aptamer sensors and ZEN aptamer sensors (Au-4MPy@AgNPs-Apt(OTA) and Au-TBN@AgNPs-Apt(ZEN) composited with rigid substrates ITO / AuNPs / GO, respectively) were used to capture, spectrally acquire, and establish standard curves for OTA standard solutions ranging from 0.001 to 20 ng / mL and ZEN standard solutions ranging from 0.1 to 150 ng / mL.

[0097] The spectrum collected by the OTA aptor sensor is as follows Figure 5c As shown, the spectrum collected by the ZEN aptamer sensor is as follows: Figure 5d As shown, the results indicate that it is located at 1005 cm. -1 4 MPy and 2227 cm -1 The characteristic peak intensities of TBN were all negatively correlated with the concentration of the standard, as shown in the standard curve results. Figure 5e , 5f As shown, the sensor used for ZEN detection exhibits no significant linearity when the standard concentration exceeds 100 ng / mL. Therefore, a standard curve was established for ZEN standard solutions ranging from 0.1 to 100 ng / mL. The characteristic peak intensities of both signal molecules are linearly negatively correlated with the logarithm of the standard concentration, and the correlation coefficients both reach R0. 2 A value above 0.99 indicates a good linear relationship.

[0098] Therefore, based on the experimental parameters of the single-probe aptamer sensor construction, the graphene concentration was set to 0.16 mg / mL, the rigid substrate immersion time to 80 min, and the AgNO3 addition amount to 1100 μL in the subsequent dual-probe aptamer sensor construction process.

[0099] Example 2:

[0100] The rigid SERS aptamer sensor prepared in Example 1 was applied to the simultaneous quantitative detection of zearalenone and ochratoxin in maize.

[0101] (1) Optimization of the ratio of the two signal probes

[0102] The ratio of signal probes to their respective targets significantly affects the sensitivity of the adsorption process on a rigid substrate. To determine the optimal ratio, a sensitivity index (ε) is introduced, where:

[0103]

[0104] I0 is the SERS signal strength of the assembled rigid SERS aptamer sensor. s The characteristic peak intensity of the SERS aptamer sensor after immersion in a mixture of OTA and ZEN standard solutions for 1 hour is the corresponding peak intensity of its respective target analytes. b This represents the background SERS signal intensity at the corresponding characteristic peak position of ITO / AuNPs / GO without signal probe assembly. ε1 and ε2 indicate the sensitivity of identifying OTA and ZEN under different signal probe assembly ratios, respectively. The more sensitive the SERS aptamer sensor, the closer its sensitivity index (ε) is to 1.

[0105] The calculated sensitivity index results are as follows Figure 6 As shown, the results indicate that when the volume ratio of the two signal probes is 5:5, their sensitivity is poor. When the volume ratio of the OTA signal probe to the ZEN signal probe is 6:4, the sensitivity indices of both probes reach their maximum values, with ε1 being 0.712 and ε2 being 0.792. Therefore, a volume ratio of 6:4 for the OTA signal probe to the ZEN signal probe is selected for the simultaneous detection of OTA and ZEN fungal toxins.

[0106] (2) Establishment of the standard curve for the mixed-signal probe SERS aptamer sensor

[0107] Based on the optimization of experimental parameters, the SERS aptamer sensor prepared in this invention was used to acquire Raman spectra of OTA and ZEN standard samples with different concentration combinations. The OTA concentrations were 0.001, 0.01, 0.1, 1, 3, 5, 10, 15, and 20 ng / mL, and the ZEN concentrations were 0.1, 1, 3, 5, 10, 20, 30, 50, and 100 ng / mL, with the two concentrations paired in ascending order. After 1 hour of capture, the samples were soaked in deionized water and gently shaken. SERS spectral data were then acquired and processed using a fully automated micro Raman spectrometer. First, a 5× low-power objective lens was used for coarse focusing until the droplet sample was clearly focused. Then, a ×10 high-power objective lens was used, and clear microscopic images were obtained by adjusting the field of view brightness and focusing.

[0108] The acquisition parameters were set as follows: excitation wavelength 638nm; excitation power 100mW; integration time 1s. Each sample was acquired 5 times.

[0109] Data Processing and Analysis: Reliable quantitative and qualitative analysis of the raw SERS spectra can effectively improve the accuracy of this detection method. LabSpec6 software was used to preprocess the raw spectra using chemometric methods to eliminate background drift caused by fluorescence absorption. Background interference was removed by baseline calibration of the spectra using polynomial background fitting, with parameters set to degree=11 and maximum points=110. The smoothing algorithm selected was the polynomial type, with parameters set to factor=9, degree=7, and size=11.

[0110] The collected spectral results are as follows Figure 7a As shown, the SERS aptamer sensor is at 1005 cm. -1 The intensity of the Raman characteristic peak at 2227 cm⁻¹ decreases with increasing OTA concentration. -1 The intensity of the Raman characteristic peak at the location decreases with increasing ZEN concentration. Figure 7b and 7c They respectively displayed 1005cm -1 The linear fitting curve between the Raman characteristic peak intensity and the lg value of OTA concentration at the specified location (0.001-20 ng / mL, y = 4556.84498-1443.70x, R0) is shown. 2 =0.993) and 2227cm -1 The linear fitting curve between the Raman characteristic peak intensity and the lg value of ZEN concentration at a given location (0.1-100 ng / mL, y = 7242.93091-2438.08x, R0) is shown. 2 =0.988), the fitting results show that the rigid SERS aptamer sensor has a good linear relationship for the detection of different concentrations of zearalenone and ochratoxin. According to 3σb / slope (σb, standard deviation of blank sample), the detection limits of this detection method for OTA and ZEN are calculated to be 0.238 ng / mL and 0.187 ng / mL, respectively.

[0111] The content of zearalenone and ochratoxin in the sample can also be further calculated based on the measured concentration. Taking zearalenone as an example, the content is calculated in the following way in this invention:

[0112] The zearalenone content in actual positive maize samples is calculated according to formula (1):

[0113]

[0114] X: The content of zearalenone in the sample, in micrograms per kilogram (μg / kg).

[0115] P: Concentration of zearalenone in the sample solution, expressed in nanograms per milliliter (ng / mL).

[0116] V: Final volume of the test sample solution, in milliliters (mL).

[0117] M: Sample weight, in grams (g)

[0118] According to the above formula, the linear range for quantification of zearalenone in this invention is 0.2-200 μg / kg, with a detection limit of 0.374 μg / kg, and the linear range for quantification of ochratoxin is 0.002-40 μg / kg, with a detection limit of 0.476 μg / kg.

[0119] Example 3:

[0120] (1) The rigid SERS aptamer sensor of the present invention is used to detect spiked corn samples.

[0121] This embodiment uses the addition of standards to corn samples that do not contain zearalenone toxin and ochratoxin to verify the practical applicability of the detection method.

[0122] The specific method is as follows:

[0123] The constructed rigid SERS aptamer sensor was used to detect single-spiked samples (OTA: 0.046, 0.60, 2.0, 4.50, 5.0, 8.0, 18 μg / kg; ZEN: 0.75, 2.50, 5.60, 13.40, 21, 37.40, 85 μg / kg) and mixed-spiked sample combinations (OTA, ZEN: 0.046, 2.50, 0.60, 5.60, 2.0, 0.75, 4.50, 21, 5.0, 37.40, 8.0, 13.40, 18, 85 μg / kg). The rigid SERS aptamer sensor assembly was immersed in the extraction solutions of single-spiked OTA and ZEN samples, as well as the mixed-spiked extraction solutions.

[0124] After capture, Raman spectra were acquired, and a standard curve was invoked to determine the contents of zearalenone and ochratoxin in the actual corn sample. The acquisition parameter settings and data processing analysis methods were the same as those used for establishing the standard curve. Based on the acquired spectral intensity, the standard curve was invoked to determine the concentrations of zearalenone and ochratoxin in the test sample.

[0125] The detection results of SERS aptamer sensors with two signal probes for single-spiked and mixed-spiked samples are as follows: Figure 7d and 7eAs shown, based on 1005cm -1 And 2227cm -1 The Raman detection values ​​of OTA and ZEN in the mixed spiked sample were calculated based on the Raman characteristic peak intensity and linear relationship at the location.

[0126] The contents of zearalenone and ochratoxin in spiked corn samples can also be calculated according to formula (1). Table 1 shows a comparison of the detection values ​​and recoveries determined by the method of this invention with those obtained by liquid chromatography. The recoveries of OTA spiked samples ranged from 92.90% to 106.60%, and the recoveries of ZEN spiked samples ranged from 93.13% to 118.38%, indicating that the SERS detection results are relatively accurate and reliable. This analytical method has high accuracy and stability for toxin detection.

[0127] Table 1. Results of simultaneous detection of mixed spiked corn samples by the method of the present invention and liquid chromatography.

[0128]

[0129]

[0130] Example 4:

[0131] This embodiment uses spiked detection of different types of toxins on corn samples to verify the specificity of the detection method.

[0132] The specificity of the constructed rigid SERS aptamer sensor was evaluated using aflatoxin B1 (AFB1), vomitoxin (DON), and fumonisin B1 (FB1) as interfering agents. Two g spiked corn samples were prepared, containing AFB1 (100 μg / kg), DON (100 μg / kg), FB1 (100 μg / kg), and a mixed sample containing AFB1, DON, FB1 (all 100 μg / kg), ZEN (20, 40 μg / kg), and OTA (2, 5 μg / kg). These samples were analyzed to assess whether other mycotoxins would affect the specificity of the SERS aptamer sensor for ZEN and OTA. The detection method for specificity analysis was the same as that used to establish the standard curve.

[0133] The specific detection results of the rigid SERS aptamer sensor for the two signal probes are as follows: Figure 7f As shown, the target mycotoxin is only present in the sample at a concentration of 1005 cm⁻¹. -1 and 2227cm -1 The intensity of the characteristic peak at the specified location will decrease; otherwise, the Raman response will remain at a high level. Therefore, it can be demonstrated that the synthesized rigid SERS aptamer sensor has good specificity for zearalenone and ochratoxin.

[0134] In summary, the SERS aptamer sensor designed in this invention, based on a modified rigid SERS-enhanced substrate, is a portable SERS sensor with good stability and environmental friendliness, capable of simultaneously and rapidly detecting the content of zearalenone and ochratoxin in maize. Utilizing the π-π covalent bond between graphene and the aptamer, as well as the biological specificity of the aptamer, interference from other substances and fungal toxins in complex samples can be eliminated, enabling simultaneous and specific detection of ochratoxin and zearalenone in maize. Subsequent calculations establish correlations between the peak values ​​at the characteristic Raman shifts of 4-mercaptopyridine (4-MPy) and 4-mercaptobenzonitrile (TBN) and the contents of ochratoxin and zearalenone, respectively, achieving highly sensitive quantitative detection of both toxins simultaneously without cumbersome pretreatment steps.

[0135] 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 with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for fabricating a rigid SERS aptamer sensor, characterized in that, The method includes the following steps: Fabrication of rigid SERS-reinforced substrates ITO / AuNPs / GO: Conductive glass ITO was ultrasonically dried in acetone, ethanol and ultrapure water respectively. Gold nanoparticles AuNPs were deposited on the ITO surface using HAuCl4 aqueous solution as electrolyte to prepare ITO / AuNPs. Then, graphene oxide solution was dropped onto the surface of the ITO / AuNPs and dried to obtain a rigid SERS-reinforced substrate ITO / AuNPs / GO. The electrodeposition conditions were as follows: electrodeposition at a potential of -1.5V for 200s; In the process of preparing the rigid SERS-reinforced substrate ITO / AuNPs / GO, the concentration of the HAuCl4 solution is 10 mM, and the concentration of the graphene oxide solution is 0.00625-0.25 mg / mL; Preparation of specific SERS probes for ochratoxin Au-4MPy@AgNPs-Apt and specific SERS probes for zearalenone Au-TBN@AgNPs-Apt: Alcoholic solutions of 4MPy and TBN were added to gold nanoparticle solutions, respectively. After adsorption of signal molecules, Au-4MPy and Au-TBN were obtained with adsorbed signal molecules 4MPy and TBN. Raman signal probes Au-4MPy@AgNPs and Au-TBN@AgNPs were prepared by adding trisodium citrate solution, AgNO3 solution, and ascorbic acid solution to Au-4MPy and Au-TBN solutions respectively and stirring. Take the aptamer solutions of OTA and ZEN, and activate them with TCEP aqueous solution. Add the activated aptamer solutions to the Raman signal probes Au-4MPy@AgNPs and Au-TBN@AgNPs respectively and incubate at room temperature. After incubation, centrifuge to remove unconnected aptamers and resuspend them in Tris-HCl buffer. Fabrication of rigid SERS aptamer sensors: The probes Au-4MPy@AgNPs-Apt and Au-TBN@AgNPs-Apt were mixed and attached to the ITO / AuNPs / GO surface to form a rigid SERS aptamer sensor. The volume ratio of the Au-4MPy@AgNPs-Apt and Au-TBN@AgNPs-Apt probes is 3.5-6.5:6.5-3.5; The aptamers include the aptamer ZEN-Apt of zearalenone and the aptamer OTA-Apt of ochratoxin. The ZEN-Apt sequence is as follows: SH-5'-AGCAGCACAGAGGTCAGATGTCATTCTATGGTACATTACTATTCCTG TAATGTGATATGCCTATGCGTGCTACCGTGAA-3', with a thiol group modified at the 5' end; The OTA-Apt sequence is as follows: SH-5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3', with a thiol group modified at the 5' end.

2. The method for fabricating a rigid SERS aptamer sensor according to claim 1, characterized in that, In the preparation of the rigid SERS-enhanced substrate ITO / AuNPs / GO, the concentration of the graphene oxide solution was 0.16 mg / mL.

3. The method for fabricating a rigid SERS aptamer sensor according to claim 1, characterized in that, After adding alcoholic solutions of 4MPy and TBN to the gold nanoparticle solution, the final concentrations of the signal molecules 4MPy and TBN in the mixed solution were both 10μM; the adsorption time of the signal molecules was 2h.

4. The method for fabricating a rigid SERS aptamer sensor according to claim 1, characterized in that, The volume ratio of the solution containing the Raman signal molecules, the trisodium citrate solution, the AgNO3 solution, and the ascorbic acid solution is 5:0.8:0.8-1.4:

2. The concentration of the trisodium citrate solution is 8.8 mM, the concentration of the AgNO3 solution is 38.8 mM, and the concentration of the ascorbic acid solution is 10 mM.

5. The method for fabricating a rigid SERS aptamer sensor according to claim 4, characterized in that, The volume ratio of the solution containing the Raman signal molecules, the trisodium citrate solution, the AgNO3 solution, and the ascorbic acid solution is 5:0.8:1.1:

2.

6. The method for fabricating a rigid SERS aptamer sensor according to claim 1, characterized in that, The molar ratio of the aptamer solution to the TCEP solution is 1:

100. The volume ratio of the aptamer solution to the Raman signal probe solution is 0.1:

1. The concentration of the aptamer solution is 10 μM. The activated aptamer solution was added to the Raman signal probes Au-4MPy@AgNPs and Au-TBN@AgNPs respectively and incubated at room temperature for 12 h; the Tris-HCl buffer is a mixture of Tris-HCl, NaCl and KCl.

7. The method for fabricating a rigid SERS aptamer sensor according to claim 1, characterized in that, The volume ratio of the Au-4MPy@AgNPs-Apt and Au-TBN@AgNPs-Apt probes is 6:

4.

8. The rigid SERS aptamer sensor prepared by the preparation method according to any one of claims 1-7, characterized in that, The rigid SERS aptamer sensor adsorbs signal molecules 4Mpy and TBN, and can simultaneously detect zearalenone and ochratoxin.

9. A method for simultaneously detecting zearalenone and ochratoxin, characterized in that, The method is based on the rigid SERS aptamer sensor of claim 8 or the rigid SERS aptamer sensor prepared by the method of any one of claims 1-7, and the method includes the following steps: S1. The prepared rigid SERS aptamer sensor was immersed in a mixed solution of standard OTA and standard ZEN. After the capture reaction, Raman spectra were collected. S2. Establish a standard curve based on the relationship between the mixed solution of standard OTA and ZEN and the characteristic Raman peaks of their corresponding signal molecules 4Mpy and TBN; S3. Detect the content of zearalenone and ochratoxin in the sample to be tested: Mix the sample to be tested with a rigid SERS aptamer sensor, collect Raman spectra according to the method in step S1, and calculate the concentration of zearalenone and ochratoxin in the sample to be tested according to the standard curve in step S2.

10. The application of the rigid SERS aptamer sensor of claim 8, or the rigid SERS aptamer sensor prepared by the method of any one of claims 1-7, or the detection method of claim 9, in the simultaneous detection of zearalenone and ochratoxin in food.

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