Double-signal aptamer sensor and preparation method and application thereof

Abstract

The invention relates to a double-signal aptamer sensor and a preparation method and application thereof, and belongs to the technical field of sensors. The dual-signal aptamer sensor comprises a first compound, a second compound and a third compound, wherein the first compound comprises Au dimers and functionalized cDNA (complementary deoxyribonucleic acid) modified on the Au dimers; the first compound is Fe3O4 (at) SiO2-CDs, and the second compound is Fe3O4 (at) SiO2-CDs and an aptamer apt of aflatoxin B1, wherein the aptamer apt of aflatoxin B1 is modified on the Fe3O4 (at) SiO2-CDs; the Fe3O4 (at) SiO2-CDs comprise Fe3O4 (at) SiO2 and CDs which are modified on the Fe3O4 (at) SiO2; the first compound and the second compound are connected through complementary base pairing to form an assembly. Due to the introduction of the Raman signal probe, the Raman signal of the assembly is enhanced, meanwhile, the Au dimers have a quenching effect on the fluorescence of the magnetic substrate, and the fluorescence signal of the assembly is weakened. After the target is added, the aptamer is preferentially specifically combined with the target, the assembly is disassembled, the Au dimers fall off from the Fe3O4 (at) SiO2-CDs, and through magnetic separation, a Raman signal of precipitation is reduced along with the addition of the target, and a fluorescence signal is recovered. The dual-mode detection is realized by measuring the change of Raman and fluorescence signals of the precipitation part along with the target addition amount.

Description

Technical Field

[0001] The present invention belongs to the technical field of sensors, and particularly relates to a dual-signal aptamer sensor, a preparation method thereof, and an application thereof. Background Art

[0002] Aflatoxin (AFT) is a type of bifuran ring toxin released by a few filamentous fungi such as Aspergillus flavus and Aspergillus parasiticus. Approximately 20 metabolic derivatives have been discovered so far. Among them, aflatoxin B1 (AFB1) has the highest yield, the greatest toxicity, and the strongest carcinogenicity. It has been determined as a Class I carcinogen by the World Health Organization. The national food safety standard stipulates that the limit in corn and peanut products is 20.0 μg / kg. Since AFB1 is widely distributed in nature, it is easy to harm human health directly or indirectly, and it is most likely to cause harm by consuming common foods in daily diets such as contaminated milk, peanuts, and rice. Research shows that AFB1 is a conjugate of methoxy, difuran ring, coumarin, and cyclopentenone. Due to its rich unsaturated bonds, it is easy to form highly reactive epoxides through metabolic activation after being ingested by the human body, affecting cell mitosis, and then leading to gene mutations and cell carcinogenesis. Thus, it can be seen that AFB1 is an important factor affecting human health.

[0003] Traditional methods for detecting AFB1 mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), but most of them have defects such as cumbersome pretreatment steps, high costs, and the proficiency of staff. Immunological assays such as enzyme-linked immunosorbent assay (ELISA) utilize the specific adsorption of antibodies and antigens, and are prone to problems such as false positive results and difficulties in preparing small molecule antibodies. Therefore, it is of great practical significance to develop a rapid, efficient, and low-cost AFB1 detection method.

[0004] In recent years, due to its advantages such as high sensitivity, rich fingerprint spectrum information, low cost, and non-destructiveness, surface-enhanced Raman scattering (SERS) technology has become a rapidly developing technology in the field of trace detection. SERS generally uses nanoscale noble metals (such as gold, silver, etc.) as active substrates, and through two mechanisms of electromagnetic field enhancement and chemical enhancement, enhances the Raman scattering signal of molecules, thereby realizing the efficient detection of trace substances. Among them, electromagnetic field enhancement occurs when incident light irradiates the surface of nanoscale metals, which will excite the local surface plasmon resonance (LSPR) on the metal surface, and then form "hot spots" in the gaps or tips of nanostructures. The Raman signals of molecules located in these regions are significantly amplified; chemical enhancement is that after the molecule adsorbs on the metal surface, charge transfer occurs or new electronic states are formed, resulting in a change in the molecular polarizability, thus enhancing the Raman scattering cross-section. Currently, functionalized SERS active substrates applied to the detection of AFB1 have also been developed to a certain extent. For example, Chen et al. (Chen Y, Chen YY, Yang YL, et al. Development of an ultrasensitive SERS aptasensor for determination of aflatoxin B1 by modifying magnetic beads with UiO-66-NH2 for enhanced signal probe capturing [J]. Sensors and Actuators: B. Chemical, 2023, 393: 134329.) synthesized a silver-coated gold (Au@4-MBA@AgNPs) sandwich structure, and the detection limit for AFB1 was 1.47×10 -7ng / mL; Chen et al. (Chen QS, Jiao TH, Yang MX, et al. Pre etched Ag nanocluster as SERS substrate for the rapid quantification of AFB1 in peanut oil via DFT coupled multivariate calibration[J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2020, 239: 118411.) used functionalized pre-etched silver nanoclusters as SERS active substrates to achieve the quantitative detection of AFB1 by the direct method, with a detection limit of 5.0 ng / mL. However, there are still the following two problems in the current SERS-based AFB1 detection: (1) For the direct detection method, the structure of AFB1 is easily affected by the complex matrix and substrate environment, and the characteristic peaks are prone to shift; (2) For the indirect detection method, the SERS substrate is unstable, the nanoparticles are prone to agglomeration, and the SERS signal enhancement effect of the substrate needs to be improved.

[0005] Fluorescence is a process in which short-wave energy is absorbed and light is emitted when returning to the ground state. When light irradiates a molecule, some electrons transition from the ground state to the excited state, and then return to the ground state and release photons. The light energy released during this process is fluorescence, and the technique of using fluorescence signals to detect and identify specific molecules or ions is called fluorescence analysis technology. Fluorescence dynamic quenching (FDQ) refers to the phenomenon in which the fluorescence intensity weakens after a fluorescent molecule collides with a quencher. The quencher causes the fluorescent molecule to return to the ground state through collision, and the energy is released in the form of heat energy, resulting in a decrease in fluorescence intensity. In recent years, sensors based on fluorescence dynamic quenching have high sensitivity, strong selectivity, simple operation, and low cost, and have been widely used in the fields of biosensors, environmental monitoring, food safety, and medical diagnosis. Wang et al. (Wang CG, Zhang WH, Qian J, et al. A FRET aptasensor for sensitive detection of aflatoxin B1 based on a novel donor-acceptor pair between ZnS quantum dots and Ag nanocubes [J]. Analytical Methods, 2020, 13: 462-468.) constructed a novel aptasensor with silver nanocubes as the quencher and zinc sulfide quantum dots as the fluorescent donor, and the detection limit for AFB1 was 2.67 pg / mL. However, when there are impurities or multiple solvents in the sample, background fluorescence usually occurs, interfering with the detection of the target signal and reducing the signal-to-noise ratio.

[0006] In summary, it is still of great significance to construct a dual-signal aptasensor combining SERS and FDQ effects to detect aflatoxin B1. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a dual-signal aptamer sensor, its preparation method and application. By utilizing the nanogap between two AuNPs and using 1,2-bis(4-pyridyl)ethylene (BPE) as a Raman signal molecule, a gold dimer (Au dimers) with strong and stable Raman signals is synthesized. After modifying the nucleic acid chain, it forms an assembly with Fe3O4@SiO2@CDs-apt with fluorescence signals through the principle of base complementary pairing. Due to the introduction of the Raman signal probe, the Raman signal of the assembly is enhanced, and at the same time, Au dimers quench the fluorescence of the magnetic substrate, weakening the fluorescence signal of the assembly. After adding the target, the aptamer preferentially binds specifically to the target, the assembly disintegrates, and Au dimers fall off from Fe3O4@SiO2-CDs. Through magnetic separation, the Raman signal of the precipitate decreases with the addition of the target, and the fluorescence signal recovers. By measuring the changes in the Raman and fluorescence signals of the precipitate part with the addition amount of the target, dual-mode detection is achieved.

[0008] The first object of the present invention is to provide a dual-signal aptamer sensor, comprising:

[0009] The first complex: Au dimers, and functionalized cDNA modified on the Au dimers;

[0010] The second complex: Fe3O4@SiO2-CDs, and the aptamer apt of aflatoxin B1 modified on the Fe3O4@SiO2-CDs; the Fe3O4@SiO2-CDs includes Fe3O4@SiO2, and CDs modified on the Fe3O4@SiO2;

[0011] The first complex and the second complex are connected through base complementary pairing to form an assembly.

[0012] In an embodiment of the present invention, the nucleotide sequence of the functionalized cDNA is 5’-SH-(CH2)6-CAGAGAGACAACACGTGCCCAAC.

[0013] In an embodiment of the present invention, the nucleotide sequence of the aptamer apt of aflatoxin B1 is: 5’-NH2-(CH2)6-AGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCAGG CCCACA.

[0014] In one embodiment of the present invention, a nucleic acid strand (aptamer complementary strand) modified with -SH is connected to Au dimers through gold-sulfur bonds, enabling the formation of an assembled structure with the Fe3O4@SiO2-CDs material modified with aptamer apt, and used as a Raman signal probe for experiments; by coating SiO2 on the surface of Fe3O4, connecting CDs after modifying amino groups, the carboxyl groups on the CDs can condense with the amino groups on the aptamer, and then a capture fluorescence probe with specific recognition of aflatoxin B1 is synthesized.

[0015] The second object of the present invention is to provide a preparation method of the double-signal aptamer sensor, comprising the following steps:

[0016] S1. Incubate the functionalized cDNA solution and the Au dimers solution to obtain Au dimers-cDNA;

[0017] S2. Incubate the aptamer apt solution of aflatoxin B1 and the Fe3O4@SiO2-CDs solution under the conditions of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to obtain Fe3O4@SiO2-CDs-apt;

[0018] S3. Incubate the solution containing Au dimers-cDNA and the solution containing Fe3O4@SiO2-CDs-apt to obtain the double-signal aptamer sensor.

[0019] In one embodiment of the present invention, in S1, the concentration of the functionalized cDNA solution is 9 μmol / L - 11 μmol / L, the concentration of the Au dimers solution is 5 nmol / L - 6 nmol / L, and the volume ratio of the functionalized cDNA solution to the Au dimers solution is 4:(23 - 27).

[0020] In one embodiment of the present invention, in S2, the concentration of the aptamer apt solution of aflatoxin B1 is 9 μmol / L - 11 μmol / L, the concentration of the Fe3O4@SiO2-CDs solution is 0.4 mg / mL - 0.6 mg / mL, and the volume ratio of the aptamer apt solution of aflatoxin B1 to the Fe3O4@SiO2-CDs solution is 1:(7 - 9).

[0021] In one embodiment of the present invention, in S2, the preparation of the Fe3O4@SiO2-CDs comprises the following steps

[0022] S21. React Fe3O4@SiO2 and (3-aminopropyl)triethoxysilane in a solvent to obtain Fe3O4@SiO2-NH2; the mass ratio of Fe3O4@SiO2 to (3-aminopropyl)triethoxysilane is 15:(470 - 475);

[0023] S22. Incubate CDs with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and then react with the Fe3O4@SiO2-NH2 solution described in S21 to obtain the Fe3O4@SiO2-CDs.

[0024] In one embodiment of the present invention, in S3, the concentration of the solution containing Au dimers-cDNA is 5 nmol / L - 6 nmol / L, the concentration of the solution containing Fe3O4@SiO2-CDs-apt is 0.4 mg / mL - 0.6 mg / mL, and the volume ratio of the solution containing Au dimers-cDNA to the solution containing Fe3O4@SiO2-CDs-apt is (2.8 - 3.2):1.

[0025] The third object of the present invention is to provide an application of the double-signal aptamer sensor described above in detecting aflatoxin B1.

[0026] In one embodiment of the present invention, the concentration of the aflatoxin B1 is 0.0001 μg / kg - 100 μg / kg.

[0027] The technical solution of the present invention has the following advantages compared with the prior art:

[0028] (1) The carbon dots CDs used in the double-signal aptamer sensor of the present invention have stronger and more stable fluorescence signals compared with metal quantum dots. Connecting CDs with the magnetically separable magnetic substrate Fe3O4@SiO2 is not only beneficial for the separation from the Raman probe, but also the loading of SiO2 can effectively improve the dispersibility of the material and avoid the quenching effect of Fe3O4 on CDs.

[0029] (2) The Au dimers used in the double-signal aptamer sensor of the present invention have good stability. The nano-gap generated by the aggregation of AuNPs amplifies the Raman hot spots in the electromagnetic field. Compared with single AuNPs, the Raman signal intensity of Au dimers can be greatly improved. In addition, Au dimers serve as both a quencher for CDs, having a good fluorescence quenching effect and being used to regulate the change of fluorescence intensity, and as a Raman probe to control the Raman signal. This double-signal aptamer sensor is applicable to the fields of food safety and analytical detection.

[0030] (3) The dual-signal aptamer sensor described in the present invention is a SERS / fluorescence dual-mode nanosensor. Compared with a single signal, dual-mode detection can further enhance the reliability and accuracy of target detection, and at the same time, the detection signal can be selected based on the existing conditions in the laboratory.

[0031] (4) The dual-signal aptamer sensor described in the present invention shows a wide linear range (0.0001 μg / kg - 100 μg / kg) and high sensitivity (the LOD of Raman signal is 0.147 pg / mL, and the LOD of fluorescence signal is 0.066 pg / mL) for the detection of AFB1. Description of the Drawings

[0032] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to specific embodiments of the present invention in combination with the drawings, where:

[0033] Figure 1 It is the transmission electron microscopy characterization diagram (TEM) of Test Example 1 of the present invention; among them, A is AuNPs, and B is Au dimers;

[0034] Figure 2 It is the transmission electron microscopy characterization diagram (TEM) and element distribution diagram of Test Example 2 of the present invention; among them, A is the TEM diagram of Fe3O4, B is the TEM diagram of Fe3O4@SiO2, C is the TEM diagram of Fe3O4@SiO2-CDs, and D is the element distribution diagram;

[0035] Figure 3 It is the Raman and fluorescence detection results of different concentrations of AFB1 solutions in Test Example 3 of the present invention; among them, A is the Raman spectrogram, B is the corresponding standard curve, C is the fluorescence spectrogram, and D is the corresponding standard curve;

[0036] Figure 4 It is the Raman and fluorescence detection results of different types of toxins in Test Example 6 of the present invention; among them, A is the Raman intensity and B is the fluorescence intensity. Detailed Embodiments

[0037] The following further describes the present invention in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. It should be understood that the specific embodiments are only used to explain the present invention, but the exemplified embodiments do not limit the present invention.

[0038] In the present invention, unless otherwise specified, the technical and scientific terms used in the present invention have the same meanings as those generally understood by those skilled in the technical field to which the present invention belongs.

[0039] In the present invention, unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods without special instructions. The materials, reagents, etc. used can be obtained from commercial channels without special instructions.

[0040] In the present invention, unless otherwise specified, the nucleotide sequence of the SH-cDNA used in the embodiments of the present invention is: 5'-SH-(CH2)6-CAGAGAGACAACACGTGCCCAAC.

[0041] In the present invention, unless otherwise specified, the nucleotide sequence of the AFB1apt used in the embodiments of the present invention is: 5'-NH2-(CH2)6-AGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCAGGCCCACA.

[0042] Example 1

[0043] The dual-signal aptamer sensor and its preparation method in this example specifically include the following steps:

[0044] S1. Synthesis of Au dimers-cDNA

[0045] S11. Synthesis of AuNPs: Heat 100 mL of 1 mM HAuCl4·3H2O solution to boiling, quickly add 15 mL of 1% trisodium citrate solution and stir vigorously. After reacting for 10 min, the color of the solution changes from light yellow to wine red. After terminating the reaction and cooling to room temperature, a seed solution of gold is obtained. Centrifuge at 10,000 rpm for 10 min to remove excess reactants, and resuspend the precipitate in ultrapure water to obtain an AuNPs solution with a concentration of 11.26 nmol / L, which is stored in the dark at 4°C for later use;

[0046] S12. Synthesis of Au dimers: Take 1 mL of AuNPs solution and add 8 μL of 1 mM 1,2-bis(4-pyridyl)ethylene (BPE) aqueous solution. After vigorously shaking for 2 min, add 12 μL of 0.05 mM AgNO3 aqueous solution to terminate the self-assembly process to avoid further connection of AuNPs; Centrifuge the obtained Au dimers at 8,000 rpm for 10 min and resuspend them in TE buffer (1 M Tris-HCl, 0.5 M EDTA, pH 8) to obtain an Au dimers solution with a concentration of 5.63 nmol / L, which is stored in the dark at 4°C for later use;

[0047] S13. Synthesis of Au dimers-cDNA: Dissolve the dry powder SH-cDNA in TE buffer to prepare a SH-cDNA solution with a concentration of 10 μM, and activate it at 95 °C for 10 min to open the single strand; add 10 μL of 1 mM TCEP solution to 120 μL of SH-cDNA solution, and activate it at room temperature for 30 min to prevent the formation of intrastrand disulfide bonds; take 160 μL of the activated SH-cDNA solution and add it to 1 mL of Au dimers solution, and incubate it in the dark at 37 °C with 200 rpm for 12 h; finally, centrifuge the synthesized capture probe Audimers-cDNA at 8000 rpm for 10 min to remove the unbound SH-cDNA, resuspend the precipitate in TE buffer to obtain a Au dimers-cDNA solution with a concentration of 5.63 nmol / L, and store it in the dark at 4 °C for standby.

[0048] S2. Synthesis of Fe3O4@SiO2-CDs-apt

[0049] S21. Synthesis of Fe3O4: Add 1.5 g of FeCl3·6H2O, 1.11 g of PEG 10000 and 3 g of NaAc to 50 mL of ethylene glycol in sequence, heat and stir at 50 °C for 4 h; transfer the mixture to a polytetrafluoroethylene hydrothermal reactor, react at 200 °C for 8 h, cool to room temperature after the reaction, and the black product obtained by using an external magnetic field is Fe3O4; then wash it alternately with deionized water and ethanol for 2 - 3 times until the supernatant is completely clear, and obtain the solid powder for standby after vacuum drying at 60 °C;

[0050] S22. Synthesis of Fe3O4@SiO2-NH2: Disperse 30 mg of Fe3O4 in 56 mL of ethanol, add 14 mL of ultrapure water and 800 μL of ammonia water, and ultrasonically treat for 30 min to completely disperse the solid; add 1.2 mL of tetraethoxysilane (TEOS) solution drop by drop every 2 s - 3 s under vigorous stirring at 270 rpm, continuously stir at room temperature for 10 h, and the brown product obtained by separating with an external magnetic field is Fe3O4@SiO2, wash it alternately with ultrapure water and ethanol for 2 - 3 times, and vacuum dry it at 60 °C. Disperse 15 mg of Fe3O4@SiO2 in 3 mL of absolute ethanol, add 500 μL of (3-aminopropyl)triethoxysilane (APTES), and then continuously stir at 200 rpm and 37 °C for 12 h; the brown product obtained by separating with an external magnetic field is Fe3O4@SiO2-NH2, wash it with ethanol for 2 - 3 times, and obtain the solid powder for standby after vacuum drying at 60 °C;

[0051] S23. Synthesis of CDs: Dissolve 2 g of citric acid and 1.9 g of urea in 50 mL of water, then transfer the mixture to a polytetrafluoroethylene hydrothermal reactor and heat it at 160 °C for 4 h. The solution changes from colorless to dark green. The obtained CDs are purified in the dark for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, and the water is changed every 8 h. The purified CDs are stored in the dark at 4 °C for later use.

[0052] S24. Synthesis of Fe3O4@SiO2-CDs: Disperse 1 mg of Fe3O4@SiO2-NH2 in 1 mL of ultrapure water to prepare a dispersion with a concentration of 1 mg / mL. Dilute 4 mL of CDs to 8 mL, add 2 mL of a 40 mg / mL solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and incubate overnight at 37 °C in the dark at 200 rpm to activate the carboxyl groups on the surface of CDs. Then, take 8 mL of the CDs with activated carboxyl groups and add them to 1 mL of Fe3O4@SiO2-NH2, and incubate at 37 °C in the dark at 200 rpm for 60 min. The brown product obtained by separating with an external magnetic field is Fe3O4@SiO2-CDs. After washing 2 - 3 times with ultrapure water, it is resuspended in 2 mL of water to obtain a Fe3O4@SiO2-CDs solution with a concentration of 0.5 mg / mL, and stored in the dark at 4 °C for later use.

[0053] S25. Synthesis of Fe3O4@SiO2-CDs-apt: Dissolve the dry powder AFB1apt in TE buffer to prepare an AFB1apt solution with a concentration of 10 μM, and activate it at 95 °C for 10 min to open the single strand. Take 100 μL of the activated AFB1apt and add it to 800 μL of the Fe3O4@SiO2-CDs solution, then add 100 μL of a 40 mg / mL EDC solution and 100 μL of a 20 mg / mL N-hydroxysuccinimide (NHS) solution to promote the connection between amino and carboxyl groups, and incubate at 37 °C in the dark at 200 rpm for 12 h. The brown product obtained by separating with an external magnetic field is the magnetic substrate Fe3O4@SiO2-CDs-apt. After washing 2 - 3 times with TE buffer, it is resuspended in 800 μL of buffer to obtain a Fe3O4@SiO2-CDs-apt solution with a concentration of 0.5 mg / mL, and stored in the dark at 4 °C for later use.

[0054] S3. Synthesis of the dual-signal aptamer sensor (Au dimers-cDNA / Fe3O4@SiO2-CDs-apt):

[0055] Mix 3 mL Au dimers-cDNA solution and 1 mL Fe3O4@SiO2-CDs-apt solution in a 10 mL centrifuge tube, incubate at 200 rpm and 37°C for 2 h to allow the two materials to bind through the principle of base complementary pairing; separate with an external magnetic field until the supernatant is clear and transparent, wash with TE buffer 2-3 times to remove unbound Au dimers-cDNA; resuspend the obtained brown solid in 1 mL buffer to obtain a Au dimers-cDNA-cDNA / Fe3O4@SiO2-CDs-apt solution with a concentration of 0.5 mg / mL.

[0056] Comparative Example 1

[0057] Basically the same as Example 1, except that: Fe3O4@SiO2-CDs is replaced by MXenes, and the fluorescent group CDs is not introduced; the volume ratio of AuNPs to BPE is replaced from AuNPs:BPE (1mM) = 125:1 to AuNPs:BPE (2mM) = 800:1.

[0058] Comparative Example 2

[0059] Basically the same as Example 1, except that CDs are replaced by CdTe QDs and -CNQDs (mass ratio 1:1).

[0060] Comparative Example 3

[0061] The method is basically the same as Example 1, except that the Au dimers are replaced with Au@Ag nanospheres.

[0062] Comparative Example 4

[0063] The method is basically the same as Example 1, except that CDs are replaced with ZnS quantum dots and Fe3O4 is not introduced.

[0064] Test Example 1

[0065] Based on Example 1, AuNPs and Au dimers were characterized by transmission electron microscopy. The results are as follows Figure 1 As shown. Figure 1 It can be seen that AuNPs are uniformly spherical, and Au dimers are composed of two AuNPs nanoparticles with good dispersion.

[0066] Test Example 2

[0067] Based on Example 1, Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2-CDs were characterized by transmission electron microscopy and element distribution. The results are as follows Figure 2 As shown. Figure 2It can be seen that Fe3O4 is spherical with uniform size, the silicon layer is uniformly coated on the outer surface of Fe3O4, with a thickness of about 30 nm, and CDs are uniformly loaded on the silicon layer.

[0068] Test Example 3

[0069] Respectively incubate 20 μL of AFB1 solutions with concentrations of 0.0001 μg / kg, 0.001 μg / kg, 0.01 μg / kg, 0.1 μg / kg, 1 μg / kg, 10 μg / kg, and 100 μg / kg with 180 μL of Au dimers-cDNA-cDNA / Fe3O4@SiO2-CDs-apt solution at 200 rpm and 37 °C for 2 h; after separation with an external magnetic field, wash 2 - 3 times with TE buffer, then resuspend in 180 μL of TE buffer, and measure the Raman intensity at an excitation wavelength of 785 nm, a 50× objective lens, a laser intensity of 15 mV, and an exposure time of 1 s, and measure the fluorescence intensity at an excitation wavelength of 360 nm. Use the logarithm of the AFB1 concentration as the abscissa and the Raman intensity at 1604 cm -1 divided by the fluorescence intensity at 430 nm as the ordinate to obtain a standard curve. The specific test results are as Figure 3 shown. It can be seen from Figure 3 that when the Raman signal is used as the signal source, the linear range is 0.0001 μg / kg - 100 μg / kg, and the LOD is 0.147 pg / mL; when the fluorescence signal is used as the signal source, the linear range is 0.0001 μg / kg - 100 μg / kg, and the LOD is 0.066 pg / mL, indicating that the dual-signal aptamer sensor shows a wide linear range and high sensitivity for the detection of AFB1.

[0070] Test Example 4

[0071] Compare the linear range and detection limit of the dual-signal aptamer sensor prepared in Example 1 with those of existing different types of sensors for the detection of AFB1, as shown in Table 1 specifically:

[0072] Table 1

[0073]

[0074]

[0075] As can be seen from Table 1, the dual-signal aptamer sensor of Example 1 has a wider linear range and a lower LOD. This is because the Au dimers formed by the interaction of BPE with gold nanoparticles through the "Au-N" bond via the pyridine nitrogen at both ends have nanoscale gaps, providing "hot spots" for the enhancement of Raman signals. At the same time, since the absorption spectrum of Au dimers overlaps significantly with the excitation spectrum of the prepared carbon dots, when the aptamer interacts with the complementary strand through the base complementary pairing principle, the distance between the two parts of the material gradually shortens to within 10 nm, meeting the conditions for fluorescence resonance energy transfer. Therefore, Au dimers can serve as good fluorescence quenchers while providing high-intensity Raman signals. In addition, Au dimers provide rich binding sites for the immobilization of SH-cDNA to improve the sensitivity of the dual-signal aptamer sensor. Additionally, the magnetic substrate Fe3O4@SiO2-CDs can provide a strong and stable fluorescence signal, and the surface of the loaded CDs contains abundant carboxyl functional groups, which is beneficial for the binding to amino-functionalized Fe3O4@SiO2. After embedding Fe3O4 with SiO2, it not only provides a site for the loading of CDs but also improves the dispersibility and stability of Fe3O4 in the solvent. All in all, the dual-signal aptamer sensor of the example is stable and easy to operate, providing a new idea for the detection of mycotoxins in complex food systems.

[0076] Test Example 5

[0077] The linear ranges and detection limits of the sensors prepared in Example 1 and Comparative Examples 1-4 for detecting AFB1 were compared, as shown in Table 2 specifically:

[0078] Table 2

[0079] specimen Linear range (ng / mL) LOD Example 1 <![CDATA[10 -4 -10 2 > 0.066 pg / mL Comparative Example 1 <![CDATA[10 -3 -10 2 > 0.6 pg / mL Comparative Example 2 <![CDATA[5×10 -2 -10 2 > 17 pg / mL Comparative Example 3 <![CDATA[10 -4 -10 2 > 0.40 pg / mL Comparative Example 4 <![CDATA[5×10 -3 -3×10 2 > 2.67 pg / mL

[0080] As can be seen from Table 2, the dual-signal aptamer sensor of Example 1 has a lower LOD and a wider linear range. In Comparative Example 1, since MXene was used as the Raman internal standard, the signal intensity was insufficient. When the internal standard signal is weak, it will cause an increase in the ratio fluctuation between the target signal and the internal standard, resulting in an increase in the error of quantitative analysis. In Comparative Example 2, because two kinds of quantum dots were used for surface modification and interfered with each other, one kind of quantum dot dominated, resulting in a reduction in the effective binding sites of the other kind of quantum dot and a weakening of the detection signal; at the same time, a single excitation light source may not be able to efficiently excite the two kinds of quantum dots simultaneously, resulting in a relatively low excitation efficiency of one kind of quantum dot. In Comparative Example 3, because the silver layer on the surface of the core-shell structure is prone to oxidation, resulting in the destruction of the nanostructure morphology and a reduction in the "hot spot" area, the Raman signal enhancement factor decreases. In Comparative Example 4, because the fluorescence intensity of the quantum dots used is low, it is easily affected by the background signal during detection, especially the sensitivity and accuracy for low-concentration targets are significantly deteriorated.

[0081] Test Example 6

[0082] Add 20 μL of different types of toxins (aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), ochratoxin (OTA), zearalenone toxin (ZEN), deoxynivalenol (DON), and a mixed solution of the above toxins) to 180 μL of Au dimers-cDNA-cDNA / Fe3O4@SiO2-CDs-apt solution and incubate for 2 h. After magnetic separation, the measurement is carried out according to the steps of Test Example 3, and the results are as follows Figure 4 shown. From Figure 4 it can be seen that the dual-signal aptamer sensor has good specificity for aflatoxin B1.

[0083] Test Example 7

[0084] To further evaluate the application of the dual-signal aptamer sensor in actual samples, peanuts, rice, and corn are selected as actual samples, and the detection results of aflatoxin B1 in the spiked peanut, rice, and corn samples are shown in Table 3

[0085] Table 3

[0086]

[0087] It can be seen from Table 3 that the dual-signal aptamer sensor has high accuracy in the detection of actual samples and has good practical applicability in food.

[0088] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A dual-signal aptamer sensor, characterized in that: include: The first complex: Au dimers, and functionalized cDNA modified on the Audimers; The second complex: Fe3O4@SiO2-CDs, and an aptamer apt of aflatoxin B1 modified on the Fe3O4@SiO2-CDs; the Fe3O4@SiO2-CDs includes Fe3O4@SiO2, and CDs modified on the Fe3O4@SiO2; The first complex and the second complex are connected by base complementary pairing to form an assembly.

2. The dual-signal aptamer sensor according to claim 1, characterized in that: The nucleotide sequence of the functionalized cDNA is 5'-SH-(CH2)6-CAGAGAGACAACACGTGCCCAAC.

3. The dual-signal aptamer sensor according to claim 1, characterized in that: The nucleotide sequence of the aptamer apt of aflatoxin B1 is: 5'-NH2-(CH2)6-AGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCAGGCCCACA.

4. The method for preparing the dual-signal aptasensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, incubating the functionalized cDNA solution and the Au dimers solution to obtain Au dimers-cDNA; S2, the aptamer apt solution of aflatoxin B1 and the Fe3O4@SiO2-CDs solution were incubated under the conditions of 1-ethyl-3-(3-dimethylaminopropyl)diimide hydrochloride and N-hydroxysuccinimide to obtain Fe3O4@SiO2-CDs-apt; S3. Incubate the solution containing Au dimers-cDNA and the solution containing Fe3O4@SiO2-CDs-apt to obtain the dual-signal aptamer sensor.

5. The method for preparing the dual-signal aptasensor according to claim 4, characterized in that: In S1, the concentration of the functionalized cDNA solution is 9 μmol / L-11 μmol / L, the concentration of the Au dimers solution is 5 nmol / L-6 nmol / L, and the volume ratio of the functionalized cDNA solution to the Au dimers solution is 4:(23-27).

6. The method for preparing the dual-signal aptasensor according to claim 4, characterized in that: In S2, the concentration of the aptamer apt solution of aflatoxin B1 is 9 μmol / L-11 μmol / L, the concentration of the Fe3O4@SiO2-CDs solution is 0.4 mg / mL-0.6 mg / mL, and the volume ratio of the aptamer apt solution of aflatoxin B1 to the Fe3O4@SiO2-CDs solution is 1:(7-9).

7. The method for preparing the dual-signal aptasensor according to claim 4, characterized in that: In S2, the preparation of the Fe3O4@SiO2-CDs comprises the following steps: S21, Fe3O4@SiO2 and (3-aminopropyl)triethoxysilane react in a solvent to obtain Fe3O4@SiO2-NH2; the mass ratio of Fe3O4@SiO2 and (3-aminopropyl)triethoxysilane is 15:(470-475); S22, incubate CDs with 1-ethyl-3-(3-dimethylaminopropyl)diimide hydrochloride, and then react with the Fe3O4@SiO2-NH2 solution described in S21 to obtain the Fe3O4@SiO2-CDs.

8. The method for preparing the dual-signal aptasensor according to claim 4, characterized in that: In S3, the concentration of the solution containing Au dimers-cDNA is 5nmol / L-6nmol / L, the concentration of the solution containing Fe3O4@SiO2-CDs-apt is 0.4mg / mL-0.6mg / mL, and the volume ratio of the solution containing Au dimers-cDNA and the solution containing Fe3O4@SiO2-CDs-apt is (2.8-3.2):

1.

9. Use of the dual-signal aptasensor according to any one of claims 1 to 3 in detecting aflatoxin B1.

10. The use according to claim 9, characterized in that: The concentration of aflatoxin B1 is 0.0001 μg / kg-100 μg / kg.

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