Construction method and application of photo-regulated electrochemical aptamer sensor based on functionalized graphene

Abstract

The application belongs to the technical field of biosensors, and particularly relates to a construction method of a light-regulated electrochemical aptamer sensor based on functionalized graphene and application thereof. The application designs and synthesizes g-C3N4-rGO heterojunction and rGO-AuNPs Schottky junction dual materials based on reduced graphene oxide (rGO), utilizes a photo-generated carrier transfer channel to regulate the rise and fall of an electroactive probe molecule methylene blue (MB) electrochemical signal, uses aptamer as a specific recognition element, uses a nucleic acid structure as a connecting bridge of the dual materials and a MB binding site, and constructs a light-regulated electrochemical aptamer sensor. The difference between an amplified background signal and a response signal is used to realize the improvement of analysis sensitivity. The application can be applied to high-sensitivity detection of mycotoxins, provides a new approach and a general method for sensitive monitoring of agricultural product fungal contamination, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensors, and particularly relates to a construction method of a light-regulated electrochemical aptamer sensor based on functionalized graphene and application thereof. BACKGROUND

[0002] Fungal toxins are common pathogens in agricultural products, and are small molecule secondary metabolites produced by toxin-producing fungi under certain environmental conditions. According to the source, they can be divided into Aflatoxins (AFs) and Ochratoxins (OTs) produced by Aspergillus; Fumonisins (FBs), Zearalenone (ZEN), Deoxynivalenol (DON) and T-2 toxin produced by Fusarium; Patulin (PAT) produced by Penicillium and the like. Fungal toxins have a high contamination rate in grains, and can be produced during the sowing, growth cycle, harvesting stage and subsequent storage and processing processes, are difficult to remove, are transmitted and accumulated in animals and human bodies through the food chain, and even in trace amounts have extremely strong immunosuppressive, teratogenic, carcinogenic and mutagenic toxic effects. Considering the low allowable limit, high frequency of occurrence and great toxicity of fungal toxins, it is urgent to develop sensitive and accurate analysis methods to prevent and control fungal contamination from the source.

[0003] The electrochemical aptamer sensing strategy combines the advantages of biomolecular specificity and electrochemical signal sensing, and has the characteristics of low cost, simple operation, fast response speed, high sensitivity and strong specificity, and is one of the most commonly used analysis methods for fungal toxins. However, the inevitable matrix interference will to some extent lead to the weakening of the signal of the electroactive probe molecule, and the high-sensitivity detection of trace fungal toxin contamination in complex samples is still a challenge. Traditional DNA structure-mediated high-sensitivity analysis methods can achieve sensitivity improvement through strand hybridization and strand displacement reaction, but usually involve lengthy procedures and difficulties in precise and repeatable assembly of DNA on the electrode surface, making it difficult to simultaneously obtain high sensitivity, short construction time and simple interface assembly. Therefore, developing new and efficient sensitivity amplification strategies is the key to meeting the practical application requirements of electrochemical sensing for crop mold monitoring. SUMMARY

[0004] In view of some deficiencies in the prior art, the application aims to utilize the competitive effect of photo-generated carriers of dual-element materials, and invent a light-regulated electrochemical aptamer sensor based on functionalized graphene to realize high-sensitivity quantitative detection of fungal toxins.

[0005] The application designs and synthesizes a g-C3N4-rGO heterojunction and a rGO-AuNPs Schottky junction binary material with rGO as a substrate, utilizes a photo-generated carrier transfer channel to regulate the rise and fall of an electroactive probe molecule methylene blue (MB) electrochemical signal, based on Au-S bonds and non-covalent interactions (π-π bonds) between g-C3N4 and bases, uses a nucleic acid chain as a connecting bridge of the binary material, and uses Apt as a specific recognition element to construct a light-regulated electrochemical aptamer sensing platform, so that the difference between the amplified background signal and the response signal is used to realize the improvement of the analysis sensitivity. In addition, the electrochemical aptamer sensor developed by the application can be used for high-sensitivity and high-selectivity detection of AFB1 in peanuts and peanut soil samples, and has important significance for high-sensitivity detection of mycotoxins in agricultural products.

[0006] The object of the application is achieved by the following technical solutions:

[0007] The application first provides a construction method of a light-regulated electrochemical aptamer sensor based on functionalized graphene, and the steps are as follows:

[0008] (1) Preparation of rGO-AuNPs nanomaterials: chloroauric acid (HAuCl4) solution is added to a reduced graphene oxide (rGO) dispersion liquid, and after magnetic stirring, the supernatant is removed by centrifugation, and the precipitate is washed and centrifuged with ultrapure water to obtain the washed product, i.e., the rGO-AuNPs nanocomposite material; finally, the obtained rGO-AuNPs nanocomposite material is dispersed in water again to obtain a rGO-AuNPs nanocomposite material dispersion liquid;

[0009] (2) Preparation of g-C3N4-rGO nanomaterials: melamine (C3H6N6) is mixed and ground with rGO to obtain a mixture, the mixture is calcined under a nitrogen atmosphere, and then cooled to room temperature to obtain a g-C3N4-rGO nanocomposite material; finally, the obtained g-C3N4-rGO nanocomposite material is ultrasonically dispersed in water to obtain a g-C3N4-rGO nanocomposite material dispersion liquid;

[0010] (3) Preparation of dSDNA-MB: the target aptamer is denoted as Apt; the complementary long chain of the target aptamer is denoted as Primer, and the complementary short chain of the Primer is denoted as ssDNA;

[0011] After mixing the Apt solution, the Primer solution and the ssDNA solution, heating reaction is performed, and after the reaction, the solution is cooled to room temperature to obtain an Apt-Primer-ssDNA solution; then, TCEP solution is added for activation reaction, and then methylene blue (MB) solution is added, and after incubation, a dSDNA-MB solution is obtained;

[0012] (4) polishing GCE with aluminum oxide powder, then ultrasonic treatment in anhydrous ethanol and ultrapure water in turn, drying to obtain a pretreated GCE electrode; then dispersing the rGO-AuNPs nanocomposite dispersion obtained in step (1) on the surface of the pretreated GCE electrode, and the electrode after drying treatment is denoted as rGO-AuNPs / GCE;

[0013] (5) modifying the dSDNA-MB solution prepared in step (3) on the surface of the rGO-AuNPs / GCE prepared in step (4) above for the first incubation, further modifying the MB solution for the second incubation, and then obtaining the modified electrode after rinsing and drying, which is a light-regulated electrochemical aptamer sensor based on functionalized graphene, denoted as dSDNA-MB / rGO-AuNPs / GCE;

[0014] Preferably, in step (1), the volume ratio of the HAuCl4 solution to the rGO dispersion is 1:1; the mass concentration of the HAuCl4 solution is 1%, and the concentration of the rGO dispersion is 2 mg / mL -1 ; the magnetic stirring time is 12 h; the centrifugal speed is 10,000 rpm, and the time is 15 min; and the concentration of the rGO-AuNPs nanocomposite dispersion is 1 mg / mL -1 .

[0015] Preferably, in step (2), the mass ratio of the C3H6N6 to the rGO is 24:1; the calcination is performed in a vacuum tube furnace, the heating time is 4 h, the heating rate is 2-2.3 ℃ / min, the calcination temperature is 550 ℃, and the time is 4 h; the cooling time is 4-5 h; and the concentration of the g-C3N4-rGO nanocomposite dispersion is 2 mg / mL -1 .

[0016] Preferably, in step (3), the target proxy has an aptamer, and the fungal toxin includes any one of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin M1 (AFM1), ochratoxin (OTA), fumonisin (FB1), zearalenone (ZEN), deoxynivalenol (DON), T-2 toxin, and patulin (PAT).

[0017] The concentrations of the Apt solution, the Primer solution and the ssDNA solution are all 10 μM, and the volume ratio is 1:1:1; the heating reaction temperature is 95℃, and the time is 10 min; the cooling time is 10-20 min; the volume ratio of the Apt-Primer-ssDNA solution, the TCEP solution and the methylene blue solution is 60:2:7, wherein the concentration of the TCEP solution is 10 mM, and the concentration of the MB solution is 1 mM; the activation reaction temperature is room temperature, and the reaction time is 1 h; the incubation temperature is 37℃, and the incubation time is 1 h.

[0018] Preferably, in step (4), the diameter of the GCE electrode is 3 mm; the particle size of the used aluminum oxide powder is 0.05 μm; the ultrasonic treatment time is 30 s; the modification amount of the rGO-AuNPs nanocomposite dispersion liquid is 6 μL, and the concentration is 1 mg mL -1 .

[0019] Preferably, in step (5), the modification amount of the dSDNA-MB solution is 6 μL, and the concentration is 2 μM; the first incubation temperature is 4℃, and the incubation time is 12 h; the modification amount of the MB solution is 6 μL, and the concentration is 10 μM; the second incubation temperature is 37℃, and the incubation time is 6 min.

[0020] The application also relates to a use of a functionalized graphene-based photo-regulated electrochemical aptamer sensor for detecting a target substance, which is any one of fungal toxins with aptamers, including aflatoxin B1, aflatoxin B2, aflatoxin M1, ochratoxin, fumonisin, zearalenone, deoxynivalenol, T-2 toxin and patulin.

[0021] The steps are as follows:

[0022] (1) different concentrations of target (X) standard solution are prepared and modified on the surface of a dSDNA-MB / rGO-AuNPs / GCE, and after incubation, rinsing and drying, a modified electrode is obtained, which is denoted as X / dSDNA-MB / rGO-AuNPs / GCE;

[0023] Then, a g-C3N4-rGO nanocomposite dispersion liquid is modified on the surface of the X / dSDNA-MB / rGO-AuNPs / GCE, and after incubation, rinsing and drying, a modified electrode is obtained, which is denoted as g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE;

[0024] (2) construction of a standard curve:

[0025] The g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE modified in step (1) is used as a working electrode, a saturated Ag / AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode for electrochemical detection; the working interface is irradiated by an external light source, and the electrochemical signal I MBon of the g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE is recorded MBon The standard curve is constructed by taking the I MBon signal as the ordinate and the logarithm of the concentration of the target standard solution as the abscissa, and the standard curve is denoted as Y MBon .

[0026] (3) Detection of AFB1 in an actual sample:

[0027] The target standard solution in step (1) is replaced by a sample extract, and then the g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE modified electrode is obtained according to the operation of step (1); electrochemical detection is performed according to the operation of step (2), and the corresponding I MBon value is obtained, and finally the concentration of the target substance in the sample is obtained by substituting the value into the standard curve Y MBon constructed in step (2), thereby realizing the detection of the target substance in an unknown sample.

[0028] Preferably, in step (1), the modification amount of the target standard solution is 6 μL, the concentration of the target standard solution is 0.001-20 ng / mL -1 , the incubation temperature is 37°C, and the incubation time is 40 min; the modification amount of the g-C3N4-rGO nanocomposite dispersion is 6 μL, the concentration is 2 mg / mL -1 , the incubation temperature is 37°C, and the incubation time is 80 min.

[0029] Preferably, in step (2), the electrochemical detection is recorded by an electrochemical workstation of Autolab PGSTAT 302N, the scanning voltage range is -0.4-0.1 V, the amplitude is 0.025 V, and the frequency is 37 Hz; the wavelength of the external light source is 365 nm, the power is 7 W / cm 2 , the vertical distance between the light source and the working interface is 2 cm; the test solution is a 0.1 M PBS buffer solution with pH = 7.4, and contains ascorbic acid (AA) with a final concentration of 0.1 M.

[0030] Preferably, in step (3), the specific process of obtaining the sample extraction solution is as follows: after the sample is crushed, it is soaked in a mixture of methanol and ultrapure water, the supernatant is extracted by oscillation, and then the sample extraction solution is obtained by centrifugation and filtration; the use amount ratio of the sample, methanol and ultrapure water is 5g:14mL:6mL; the oscillation extraction time is 1h; the centrifugation speed is 8000rpm, and the time is 15min; and the filter membrane pore size is 0.22μm.

[0031] The beneficial effects of the present application are as follows:

[0032] (1) The photo-regulated electrochemical aptamer sensor of the present application has the characteristics of high sensitivity, strong specificity, good selectivity and low cost, and does not require complex sensor construction steps.

[0033] (2) The traditional sensitivity regulation strategy based on nucleic acid chains relies on complex sensor interface assembly, and the regulation efficiency is limited. The present application is based on photo-electric energy conversion, utilizes the competitive effect of photo-generated carriers of binary materials, regulates the MB electrochemical redox process, provides sensitive signal changes by amplifying the difference between the background signal and the response signal, enhances the sensor analysis sensitivity by 1.33 times, simplifies the interface assembly, and has high regulation efficiency, thereby providing a powerful means for improving the sensitivity of the sensor.

[0034] (3) The photo-regulated electrochemical aptamer sensor of the present application realizes high-sensitivity quantitative detection of AFB1 in agricultural products; the sensor has a good linear relationship with the logarithmic value of the detection concentration of AFB1 in the range of 1pg mL -1 ~10ng mL -1 , and the detection limit is 0.73pg mL -1 . The sensor has a wide detection linear range, high detection sensitivity and low detection limit.

[0035] (4) By changing the type of aptamer, the photo-regulated electrochemical aptamer sensor strategy of the present application can be extended to high-sensitivity detection of other mycotoxins, and has great application potential in the monitoring field related to agricultural product safety. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 FIG. 1 is a schematic diagram of the construction of a photo-regulated electrochemical aptamer sensor based on functionalized graphene for AFB1 detection.

[0037] Figure 2 FIG. 2 is a sensor feasibility verification diagram, wherein (A) is a sensor construction process cyclic voltammogram, and (C) is a sensor construction process energy diagram.

[0038] Figure 3 FIG. 3 is a sensor electrochemical response diagram before and after light excitation of different concentrations of AFB1, wherein (A) is a sensor electrochemical response diagram before light excitation of 1×10-3 , 5 x 10 -3 , 1 x 10 -2 , 5 x 10 -2 , 1 x 10 -1 , 5 x 10 -1 , 1, 5, 10 and 20 ng mL -1 ); (B) is I MB Calibration curve of the sensor's photoelectrochemical signal response to the logarithm of AFB1 concentration.

[0039] Figure 4 (A) is the photoelectrochemical signal response of the sensor to AFM1, AFB2, AFG1, FB1, a mixture of the four toxins (AFM1+AFB2+AFG1+FB1), AFB1, and a mixture of the four toxins (AFM1+AFB2+AFG1+FB1+AFB1); (B) is the reproducibility of the parallel measurement of the 7 electrodes; (C) is the long-term stability of the sensor. DETAILED DESCRIPTION

[0040] The embodiments of the present application will be described in detail below with reference to the accompanying drawings. The embodiments are based on the technical solutions of the present application and give detailed implementation steps and specific operation processes, but the protection scope of the present application is not limited to the following embodiments.

[0041] The photo-regulated electrochemical aptamer sensor based on functionalized graphene is suitable for high-sensitivity detection of fungal toxins. By changing the type of aptamer, the corresponding fungal toxin can be detected, and the corresponding aptamer can be purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0042] The present application is specifically implemented by detecting AFB1:

[0043] Among them, the AFB1 aptamer (Apt) sequence is: 5'-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTAGGC CCA CA-3'(50mer); the long-chain primer (Primer) complementary to Apt is: 5'-SH-(CH2)6-CAACTT CTA TGT GGG CCT AGC GAA GGG CAC GAG ACA CAG AGA GAC AAC ACG TGCCCAAC-3'(59mer); and the short-chain DNA (ssDNA) complementary to Primer is: 5'-TAG AAG TTG-3'(9mer), all purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0044] Example 1:

[0045] (1) Preparation of rGO-AuNPs nanomaterials: 2 mL of 1% HAuCl4 solution by mass concentration was added to 2 mL of 2 mg mL -1 rGO dispersion solution, and magnetic stirring for 12 h, then the solution was centrifuged at 10,000 rpm for 15 min, the supernatant was removed, and the precipitate was collected and washed with ultrapure water and centrifuged 3 times to remove excess HAuCl4, to obtain rGO-AuNPs nanocomposites; finally, the obtained rGO-AuNPs nanocomposites were redispersed in 4 mL of H2O to obtain 1 mg mL -1 rGO-AuNPs nanocomposite dispersion solution, and stored at 4°C in the dark for standby;

[0046] (2) Preparation of g-C3N4-rGO nanomaterials: 1 g of melamine (C3H6N6) was mixed with 0.042 g of rGO by grinding, and the mixture was placed in an alumina crucible and calcined in a vacuum tube furnace, heated to 550°C at a heating rate of 2-2.3°C / min under a nitrogen atmosphere for 4 h, and kept at 550°C for 4 h, then cooled to room temperature for 4-5 h to obtain g-C3N4-rGO nanocomposites; finally, the obtained g-C3N4-rGO nanocomposites were ultrasonically dispersed in water to obtain a g-C3N4-rGO nanocomposite dispersion solution with a concentration of 2 mg mL -1 ;

[0047] (3) Preparation of dSDNA-MB: 10 mM Apt solution, 10 mM Primer solution, and 10 mM ssDNA solution were mixed in equal volumes, heated at 95°C for 10 min, and then cooled to room temperature for 10-20 min after reaction, to obtain an Apt-Primer-ssDNA solution by base pairing; then 20 mL of 10 mM TCEP solution was added to 600 mL of Apt-Primer-ssDNA solution and reacted at room temperature for 1 h to activate the thiol group; finally, 70 mL of 1 mM MB solution was mixed with the Apt-Primer-ssDNA solution, and incubated at 37°C for 1 h to obtain a dSDNA-MB solution;

[0048] (4) Φ = 3 mm GCE was polished to mirror gloss with 0.05 um aluminum oxide powder, and then ultrasonically treated in anhydrous ethanol and ultrapure water for 30 s, and dried in air; then 6 mL of 1 mg mL -1 rGO-AuNPs nanocomposite dispersion solution prepared in step (1) was modified to the pretreated GCE, and dried at room temperature, and the modified electrode was denoted as rGO-AuNPs / GCE;

[0049] (5) Preparation of dSDNA-MB / rGO-AuNPs / GCE: 6 μL of 2 uM dSDNA-MB solution prepared in step (3) was modified on the surface of rGO-AuNPs / GCE prepared in step (4), and incubated at 4°C for 12 h. After washing and drying, 6 μL of 10 μM MB solution was further modified to block the adsorption sites of rGO, and incubated at 37°C for 6 min. After washing and drying, the modified electrode was obtained, which was recorded as dSDNA-MB / rGO-AuNPs / GCE, namely the light-regulated electrochemical aptamer sensor based on functionalized graphene.

[0050] The use of the light-regulated electrochemical aptamer sensor based on functionalized graphene for detecting aflatoxin B1, the steps are as follows:

[0051] (1) 6 μL of AFB1 solution (concentration of 1 x 10 -3 , 5 x 10 -3 , 1 x 10 -2 , 5 x 10 -2 , 1 x 10 -1 , 5 x 10 -1 , 1, 5, 10 and 20 ng mL -1 ) was modified on the dSDNA-MB / rGO-AuNPs / GCE prepared in step (5) of Example 1, and incubated at 37°C for 40 min. After washing and drying with PBS solution (pH = 7.4), the modified electrode was obtained, which was recorded as AFB1 / dSDNA-MB / rGO-AuNPs / GCE;

[0052] (2) 6 μL of 2 mg mL -1 g-C3N4-rGO nanocomposite dispersion was modified on AFB1 / dSDNA-MB / rGO-AuNPs / GCE, and incubated at 37°C for 80 min. After washing and drying, the modified electrode was obtained, which was recorded as g-C3N4-rGO / AFB1 / dSDNA-MB / rGO-AuNPs / GCE;

[0053] (3) AFB1 / dSDNA-MB / rGO-AuNPs / GCE prepared in step (1) and g-C3N4-rGO / AFB1 / dSDNA-MB / rGO-AuNPs / GCE modified in step (2) were used as working electrodes, a saturated Ag / AgCl electrode was used as a reference electrode, and a platinum wire electrode was used as a counter electrode. An external light source was used to irradiate the working interface at a distance of 2 cm, with a wavelength of 365 nm and a power of 7 W / cm 2The electrochemical signals of AFB1 / dSDNA-MB / rGO-AuNPs / GCE before light excitation were recorded by an electrochemical workstation with model of Autolab PGSTAT 302N MBoff and g-C3N4-rGO / AFB1 / dSDNA-MB / rGO-AuNPs / GCE after light excitation IMB on ; the test was carried out in 0.1M PBS buffer solution containing 0.1M AA with final concentration, pH=7.4, the scanning voltage range was-0.4~0.1V, the amplitude was 0.025V, and the frequency was 37Hz; and I MBoff and I MBon Two signals were compared with the logarithm of AFB1 concentration to construct two standard curves to evaluate the sensitivity improvement effect; the specific linear relationship was as shown in Figure 3 (A), with the increase of AFB1 concentration, I MB gradually decreased, when there was low concentration of AFB1, I MBon was greater than I MBoff , when there was high concentration of AFB1, I MBon was less than I MBoff , thus presenting two linear relationships with different sensitivities; the specific linear relationship diagram was as shown in Figure 3 (B), in the range of 10pg mL -1 ~20ng mL -1 , the logarithm of AFB1 concentration and I MBoff presented a good linear relationship, and the linear curve was I MB(off) =-9.70lgC AFB1 +26.02(R 2 =0.998), in the range of 1pg mL -1 ~10ng mL -1 , the logarithm of AFB1 concentration and I MBon presented a good linear relationship, and the linear curve was I MB(on) =-12.89lgC AFB1 +22.76(R 2 =0.998), the introduction of light regulation made the analysis sensitivity enhanced by 1.33 times, thus I MBon signal was used for sensing detection;

[0054] In order to evaluate the selectivity of the light-regulated electrochemical aptamer sensor, interference experiments were carried out by using AFM1, AFB2, AFG1, FB1, four toxin mixtures (AFM1+AFB2+AFG1+FB1), AFB1, and five toxin mixtures (AFM1+AFB2+AFG1+FB1+AFB1). As Figure 4(A) shows, I MBon The signal response of 10 times concentration of interfering substances can be ignored compared with the signal intensity; therefore, the sensor has good selectivity for AFB1 detection. The reproducibility of the sensor was evaluated by 7 parallel measurements of AFB1 by the aptamer sensor, and the results are shown in Figure 4 (B) shows, I MBon The RSD of 2.76% indicates that the sensor has good reproducibility. The aptamer sensor was placed in a dark environment at 4°C for a long time to evaluate the long-term stability of the sensor, and the results are shown in Figure 4 (C) shows that after 7 days, I MBon still accounts for 96.74% of the initial signal value, and has a low RSD, indicating that the light-regulated aptamer sensor constructed has quite excellent long-term stability.

[0055] (4) Based on the excellent analytical performance of the sensor, actual samples of peanuts and peanut soil were collected, and AFB1 in the actual samples was analyzed;

[0056] First, the sample extract was obtained: 5 g of ground peanut sample or peanut soil was soaked in a mixed solution containing 14 mL of methanol and 6 mL of ultrapure water, and extracted by shaking for 1 h, followed by centrifugation at 8000 rpm for 15 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain the sample extract;

[0057] The AFB1 standard solution of step (1) was replaced with the sample extract, and the g-C3N4-rGO / AFB1 / dSDNA-MB / rGO-AuNPs / GCE prepared in step (2) was obtained. The corresponding I MBon value was obtained by electrochemical mode testing according to step (3), and finally the value was substituted into the standard curve constructed in step (3) to obtain the concentration of AFB1 in the sample.

[0058] The analysis results are shown in the table. The light-regulated aptamer sensor constructed was used to analyze AFB1 of different concentrations in actual samples of peanuts and peanut soil, and the recovery rates were between 99.40% and 106.20%. Compared with the national standard method HPLC-FL (95.80% ~ 100.40%), the aptamer sensor proposed has higher reliability for the analysis of AFB1 in actual samples of peanuts and peanut soil.

[0059]

[0060] Note: "ND" means not detected; the moldy soil sample was taken from the soil around the moldy peanuts.

[0061] Description: The above examples are only used to illustrate the technical solutions described in the present application and do not limit the present application; therefore, although the present application has been described in detail with reference to the above various embodiments, those of ordinary skill in the art should understand that the present application can still be modified or equivalently replaced; and all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

Claims

1. A method for constructing a photomodulated electrochemical aptamer sensor based on functionalized graphene, characterized in that, The steps are as follows: (1) Preparation of rGO-AuNPs nanomaterials: Chloroauric acid solution was added to the reduced graphene oxide dispersion, and after magnetic stirring, the supernatant was removed by centrifugation. The precipitate was collected, washed with ultrapure water, and centrifuged to obtain the washed product, which is the rGO-AuNPs nanocomposite material. Finally, the obtained rGO-AuNPs nanocomposite material was redispersed in water to obtain the rGO-AuNPs nanocomposite material dispersion. (2) Preparation of g-C3N4-rGO nanomaterials: Melamine and reduced graphene oxide were mixed and ground to obtain a mixture. The mixture was calcined under a nitrogen atmosphere and then cooled to room temperature to obtain g-C3N4-rGO nanocomposite material. Finally, the obtained g-C3N4-rGO nanocomposite material was ultrasonically dispersed in water to obtain g-C3N4-rGO nanocomposite material dispersion. (3) Preparation of dSDNA-MB: The target aptamer is denoted as Apt; the complementary long chain of the target aptamer is denoted as Primer, and the complementary short chain of Primer is denoted as ssDNA; The Apt solution, Primer solution, and ssDNA solution were mixed and heated to react. After the reaction, the mixture was cooled to room temperature to obtain the Apt-Primer-ssDNA solution. TCEP solution was then added to activate the reaction, followed by the addition of methylene blue solution. After incubation, the dSDNA-MB solution was obtained. (4) Polish the glassy carbon electrode with aluminum oxide powder, and then treat it with anhydrous ethanol and ultrapure water in sequence. After drying, the pretreated glassy carbon electrode is obtained. Then, the rGO-AuNPs nanocomposite dispersion obtained in step (1) is modified onto the surface of the pretreated glassy carbon electrode. The electrode after drying is called rGO-AuNPs / GCE. (5) Take the dSDNA-MB solution prepared in step (3) and modify it on the surface of rGO-AuNPs / GCE prepared in step (4) for the first incubation. After incubation, further modify the MB solution and incubate again. After incubation, the modified electrode obtained by rinsing and drying is the photo-regulated electrochemical aptamer sensor based on functionalized graphene, denoted as dSDNA-MB / rGO-AuNPs / GCE. (6) Target standard solutions of different concentrations were prepared and modified on the surface of dSDNA-MB / rGO-AuNPs / GCE. After incubation, rinsing and drying, the modified electrode was obtained and denoted as X / dSDNA-MB / rGO-AuNPs / GCE. Then, the g-C3N4-rGO nanocomposite dispersion was modified on the surface of X / dSDNA-MB / rGO-AuNPs / GCE. After incubation, rinsing, and drying, the modified electrode was obtained, denoted as g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE.

2. The method for constructing a photomodulated electrochemical aptamer sensor based on functionalized graphene according to claim 1, characterized in that, In step (1), the volume ratio of the HAuCl4 solution to the rGO dispersion is 1:1; wherein the mass concentration of the HAuCl4 solution is 1%, and the concentration of the rGO dispersion is 2 mg / mL. -1 The magnetic stirring time was 12 hours; the centrifugation speed was 10,000 rpm for 15 minutes; the concentration of the rGO-AuNPs nanocomposite dispersion was 1 mg / mL. -1 .

3. The method for constructing a photomodulated electrochemical aptamer sensor based on functionalized graphene according to claim 1, characterized in that, In step (2), the mass ratio of C3H6N6 to rGO is 24:1; the calcination is carried out in a vacuum tube furnace, with a heating time of 4 hours, a heating rate of 2-2.3℃ / min, a calcination temperature of 550℃, and a calcination time of 4 hours; the cooling time is 4-5 hours; and the concentration of the g-C3N4-rGO nanocomposite dispersion is 2 mg / mL. -1 .

4. The method for constructing a photomodulated electrochemical aptamer sensor based on functionalized graphene according to claim 1, characterized in that, In step (3), the target refers to fungal toxins with aptamers, including any one of aflatoxin B1, aflatoxin B2, aflatoxin M1, ochratoxin, fumonisin, zearalenone, deoxynivalenol, T-2 toxin, and patulin. The concentrations of the Apt solution, Primer solution, and ssDNA solution were all 10 μM, with a volume ratio of 1:1:

1. The heating reaction was carried out at 95°C for 10 min, followed by a cooling time of 10–20 min. The volume ratio of the Apt-Primer-ssDNA solution, TCEP solution, and methylene blue solution was 60:2:7, with the TCEP solution having a concentration of 10 mM and the MB solution having a concentration of 1 mM. The activation reaction was carried out at room temperature for 1 h, and the incubation was carried out at 37°C for 1 h.

5. The method for constructing a photomodulated electrochemical aptamer sensor based on functionalized graphene according to claim 1, characterized in that, In step (4), the diameter of the GCE electrode is 3 mm; the particle size of the alumina powder used is 0.05 μm; the ultrasonic treatment time is 30 s; the modification amount of the rGO-AuNPs nanocomposite dispersion is 6 μL, and the concentration is 1 mg / mL. -1 .

6. The method for constructing a photomodulated electrochemical aptamer sensor based on functionalized graphene according to claim 1, characterized in that, In step (5), the modification amount of the dSDNA-MB solution is 6 μL, the concentration is 2 μM, the temperature of the first incubation is 4℃, and the incubation time is 12 h; the modification amount of the MB solution is 6 μL, the concentration is 10 μM, and the temperature of the second incubation is 37℃, and the time is 6 min; in step (6), the modification amount of the target standard solution is 6 μL, and the concentration of the target standard solution is 0.001~20 ng / mL. -1 The incubation temperature was 37℃, and the incubation time was 40 min; the modification amount of the g-C3N4-rGO nanocomposite dispersion was 6 μL, and the concentration was 2 mg / mL. -1 The incubation temperature was 37℃ and the incubation time was 80 minutes.

7. The use of the photomodulated electrochemical aptamer sensor based on functionalized graphene prepared according to any one of claims 1-6 for detecting target analytes, characterized in that, The target is a fungal toxin with an aptamer, including any one of aflatoxin B1, aflatoxin B2, aflatoxin M1, ochratoxin, fumonisin, zearalenone, deoxynivalenol, T-2 toxin, and patulin. The specific steps of the detection are as follows: (1) Construction of the standard curve: Using the modified g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode, electrochemical detection was performed. The working interface was illuminated by an external light source, and the electrochemical signal of g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE was acquired and recorded as I. MBon ; with I MBon A standard curve was constructed by comparing the concentration of the target standard solution with the logarithm of the target standard solution concentration. The resulting standard curve is denoted as Y. MBon ; (2) Detection of AFB1 in actual samples: The target standard solution was replaced with the sample extraction solution, which was then used to modify the surface of dSDNA-MB / rGO-AuNPs / GCE. After incubation, rinsing, and drying, a modified electrode was obtained, denoted as X / dSDNA-MB / rGO-AuNPs / GCE. Then, the g-C3N4-rGO nanocomposite dispersion was used to modify the surface of X / dSDNA-MB / rGO-AuNPs / GCE. After incubation, rinsing, and drying, a modified electrode was obtained, denoted as g-C3N4-rGO / X / dSDNA-MB / rGO-AuNPs / GCE. Electrochemical detection was then performed according to step (1) to obtain the corresponding I. MBon The value is then substituted into the standard curve Y constructed in step (1). MBon This allows us to determine the concentration of the target substance in the sample, thus enabling the detection of the target substance in an unknown sample.

8. The use according to claim 7, characterized in that, In step (2), the modification amount of the sample extract is 6 μL; the incubation temperature is 37℃ and the incubation time is 40 min; the modification amount of the g-C3N4-rGO nanocomposite dispersion is 6 μL and the concentration is 2 mg / mL. -1 The incubation temperature was 37℃ and the incubation time was 80 minutes.

9. The use according to claim 7, characterized in that, In step (2), the electrochemical detection was recorded by an Autolab PGSTAT 302N electrochemical workstation with a scanning voltage range of -0.4 to 0.1 V, an amplitude of 0.025 V, and a frequency of 37 Hz; the external light source had a wavelength of 365 nm and a power of 7 W / cm². 2 The vertical distance between the light source and the working interface is 2 cm; the test solution is 0.1 M PBS buffer with pH = 7.4, containing ascorbic acid at a final concentration of 0.1 M.

10. The use according to claim 7, characterized in that, In step (2), the specific process for obtaining the sample extract is as follows: after the sample is crushed, it is soaked in a mixture of methanol and ultrapure water, the supernatant is extracted by shaking, and then centrifugation and filtration are performed to obtain the sample extract; the ratio of sample, methanol and ultrapure water is 5g:14mL:6mL; the shaking extraction time is 1h; the centrifugation speed is 8000rpm and the time is 15min; the filter membrane pore size is 0.22μm.

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