Construction method and application of colorimetric and light-enhanced electrochemical dual-mode aptasensor based on bifunctional probe

Abstract

The present application belongs to the technical field of biosensor, and particularly relates to a construction method of a colorimetric-light enhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe and application thereof. The present application obtains a double signal channel of a probe methylene blue (MB) by using a streptavidin magnetic bead, and obtains supernatant with color recognition for colorimetric analysis through specific release of MB at the interface of the magnetic bead. The electron transfer path of a reduced graphene oxide-gold nanometer Schottky junction is studied, and the photo-generated electron promotes the redox process of MB, so that electrochemical signal amplification is realized. Higher response sensitivity can be obtained based on light excitation signal amplification for AFB1 trace analysis, and self-checking can be performed through I MB signals before and after light excitation to improve the reliability and accuracy of detection. Through rapid qualitative and accurate quantitative analysis of AFB1, early warning and accurate monitoring of agricultural sample mildew analysis can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe and its application. Background Technology

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus, and is a common pathogen in agricultural products. It is highly toxic and carcinogenic, and can cause acute hepatitis and hemorrhagic necrosis. Even low-level exposure can adversely affect growth and immune resistance. Therefore, there is a need to develop high-performance sensors for rapid and accurate detection of AFB1.

[0003] Electrochemical sensors have attracted widespread attention due to their advantages such as fast response, high sensitivity and strong specificity. However, the practical application of electrochemical sensors in AFB1 mold monitoring is still challenging: (1) Inevitable matrix interference may reduce the electrochemical signal of probe molecules, limiting the ultrasensitive detection of AFB1 trace contamination in complex sample matrices. At present, nucleic acid chain amplification technology is often used for signal amplification, which can effectively improve the response sensitivity, but such methods are time-consuming and inhibit the efficiency of sensor construction. Therefore, it is urgent to develop new signal amplification strategies; (2) Compared with the visual analysis of colorimetric sensors, the relatively complex readout mode of electrochemical sensors limits their application in on-site detection. The design of dual-mode sensing method allows the sensing platform to retain the distinct advantages of each mode and compensate for each other's shortcomings, which can effectively improve the analytical performance. The introduction of multiple indicator probes can effectively complete the construction of dual-mode sensing platform, but the relatively cumbersome assembly, such as deposition, etching and enzyme-catalyzed chromogenic substrate treatment, limits its practical application to some extent. Therefore, it is urgent to develop a simple and efficient multifunctional probe indicator strategy with dual signal response. Summary of the Invention

[0004] This paper aims to develop a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on the bifunctional probe methylene blue (MB) by utilizing photogenerated electron transfer in Schottky junctions, enabling rapid and accurate detection of AFB1.

[0005] This invention utilizes streptavidin magnetic beads to obtain a dual-signal channel for the probe MB. Through the specific release of MB at the magnetic bead interface, a color-identifiable supernatant is obtained for colorimetric analysis. The electron transfer pathway of the reduced graphene oxide-gold nanoparticle (rGO-AuNPs) Schottky junction was investigated, and photogenerated electrons were used to promote the redox process of MB, thereby amplifying the electrochemical signal. Furthermore, the dual-mode aptamer sensor developed in this invention can perform highly sensitive and selective detection of AFB1 in peanut and peanut soil samples, providing a powerful tool for mold analysis of agricultural samples.

[0006] The objective of this invention is achieved through the following technical solutions:

[0007] This invention first provides a method for fabricating a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor, the steps of which are as follows:

[0008] (1) Preparation of rGO-AuNPs nanomaterials: Chloroauric acid (HAuCl4) solution was added to a reduced graphene oxide (rGO) dispersion, and after magnetic stirring, the supernatant was removed by centrifugation. The precipitate was collected, washed with ultrapure water, and centrifuged to obtain rGO-AuNPs nanocomposite material. Finally, the obtained rGO-AuNPs nanocomposite material was redispersed in water to obtain an rGO-AuNPs nanocomposite material dispersion. Preferably, in step (1), the volume ratio of HAuCl4 solution to rGO dispersion is 1:1; wherein the mass concentration of HAuCl4 solution is 1%, and the concentration of rGO dispersion is 2 mg / mL. -1 The magnetic stirring time was 12 h; the centrifugation speed was 10000 rpm for 15 min; and the concentration of the rGO-AuNPs nanocomposite dispersion was 2 mg / mL. -1 .

[0009] (2) Pretreatment of streptavidin magnetic beads (SMBs): After magnetic separation, the streptavidin magnetic beads are removed from the protective solution. The precipitate is collected, washed with buffer solution, and then magnetically separated again to obtain SMBs precipitate. Finally, the SMBs precipitate is redispersed in buffer solution and resuspended by shaking to obtain an SMBs suspension. Preferably, in step (2), the amount of streptavidin magnetic beads used is 100 μL and the concentration is 10 mg / mL. -1 The buffer solution is Tris-HCl buffer, pH = 7.4, and the volume of buffer used for each wash is 1 mL; the concentration of the SMBs suspension is 2–3 mg / mL. -1 .

[0010] (3) Preparation of Apt-Primer-MB: The AFB1 aptamer solution (denoted as Apt) and the complementary chain solution of AFB1 aptamer (denoted as Primer) are mixed and heated to react. After the reaction, the mixture is cooled to room temperature to obtain the Apt-Primer solution. Then, the methylene blue (MB) solution is mixed with the Apt-Primer solution and incubated to obtain the Apt-Primer-MB solution. Preferably, in step (3), the concentrations of the Apt solution and the Primer solution are both 10 μM and the volume ratio is 1:1. The heating temperature is 95 °C and the time is 10 min. The cooling time is 10 to 20 min. The volume ratio of the MB solution to the Apt-Primer solution is 45 μL: 240 μL, where the concentration of the MB solution is 1 mM, the incubation temperature is 37 °C, and the incubation time is 1 h.

[0011] (4) Preparation of Apt-Primer-MB SMBs: Add Apt-Primer-MB solution to the SMBs suspension prepared in step (2), mix by rotation and then perform magnetic separation to obtain the precipitate, which is denoted as Apt-Primer-MB SMBs.

[0012] Preferably, in step (4), the volume ratio of the SMBs suspension to the Apt-Primer-MB solution is 9:1; the temperature of the mixing reaction is 30 °C and the reaction time is 75 min.

[0013] This invention also relates to the use of a colorimetric-photoexcited electrochemical dual-mode aptamer sensor based on a bifunctional probe for detecting AFB1, the steps of which are as follows:

[0014] (1) Pretreatment of glassy carbon electrode (GCE): GCE is polished with aluminum oxide powder, and then ultrasonically treated in anhydrous ethanol and ultrapure water in sequence. After drying, the pretreated GCE electrode is obtained.

[0015] (2) The rGO-AuNPs nanocomposite dispersion was modified onto the surface of the GCE electrode pretreated in step (1), and the electrode after drying was denoted as rGO-AuNPs / GCE;

[0016] Preferably, in step (1), the diameter of the GCE electrode is 3 mm; the particle size of the aluminum oxide powder used is 0.05 μm; and the ultrasonic treatment time is 30 s.

[0017] Preferably, in step (2), the modification amount of the rGO-AuNPs nanocomposite dispersion is 6 μL, and the concentration is 2 mg / mL. -1 .

[0018] (3) Prepare AFB1 standard solutions of different concentrations and mix them with the prepared Apt-Primer-MB SMBs for incubation. After incubation, collect the supernatant by magnetic separation to obtain supernatant at different concentrations.

[0019] Preferably, in step (3), the volume ratio of the Apt-Primer-MB SMBs precipitate to the AFB1 standard solution is 1 mg: 100 μL, and the concentration of the AFB1 standard solution is 10. -3 ~10 5 ng mL -1 The incubation temperature was 37 °C and the incubation time was 40 min.

[0020] (4) Take the complementary strand solution of the primer and denot it as Padlock solution; mix Padlock solution with the supernatant prepared in step (3) and incubate. After incubation, add TCEP solution to carry out the activation reaction. After the reaction, Primer-Padlock-MB solution is obtained.

[0021] Preferably, in step (4), the volume ratio of the supernatant, Padlock solution and TCEP solution is 5:5:1, wherein the concentration of Padlock solution is 2.5 μM and the concentration of TCEP solution is 1 mM; the incubation temperature is 37 °C and the time is 1 h; the activation reaction temperature is room temperature and the reaction time is 1 h.

[0022] (5) Take the Primer-Padlock-MB solution prepared in step (4) and modify it on the surface of rGO-AuNPs / GCE prepared in step (2) above. After incubation, immerse the modified electrode in MB solution. The modified electrode after immersion is called Primer-Padlock-MB / rGO-AuNPs / GCE.

[0023] Preferably, in step (5), the amount of Primer-Padlock-MB solution used for modification is 6 μL, the incubation temperature is 4℃, and the incubation time is 12 h; the amount of MB solution used is 200 μL, the concentration is 5 μM, and the soaking time is 2 min.

[0024] (6) Construction of the standard curve:

[0025] (a) Colorimetric mode: Using the supernatant prepared in step (3) as the test object, the RGB values ​​were obtained and recorded, and the total color difference value ΔC was calculated according to the Euclidean distance formula. A standard curve was constructed using the colorimetric signal ΔC and the logarithm of the corresponding AFB1 concentration;

[0026] (b) Electrochemical mode: Using the modified Primer-Padlock-MB / rGO-AuNPs / GCE from step (5) as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, electrochemical detection was performed; the working interface was irradiated with an external light source, and the electrochemical signals before and after photoexcitation were recorded as I0, ... MBoff and I MBon ; with I MBoff and I MBon Two standard curves were constructed by comparing the two signals with the logarithm of AFB1 concentration.

[0027] Preferably, in step (6) (a), the colorimetric determination is performed by recording and detecting RGB values ​​using a CS-420 spectrophotometer with an aperture of 8 mm, a light source type of D65, and a light source angle of 10°.

[0028] In step (6) (b), the electrochemical detection is recorded and acquired by an Autolab PGSTAT 302N electrochemical workstation with a scanning voltage range of 0 to -0.4 V, an amplitude of 0.025 V, and a frequency of 37 Hz; the light source has 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 (AA) at a final concentration of 0.1 M.

[0029] (7) Detection of AFB1 in actual samples:

[0030] (a) First, obtain the sample extract, replace the AFB1 standard solution in step (3) with the sample extract, and then operate according to the colorimetric mode in step (6) (a) to test and obtain the corresponding ΔC value; substitute the value into the standard curve constructed in step (6) (a) to obtain the concentration of AFB1 in the sample and realize the detection of AFB1 in unknown samples.

[0031] (b) After replacing the AFB1 standard solution in step (3) with the sample extract to obtain the supernatant, follow the steps (4) and (5) to obtain the modified electrode after soaking; then follow the electrochemical mode in step (6) (b) to test and obtain the corresponding I MBoff I MBon The value is then substituted into the standard curve constructed in step (b) of step (6) to obtain the concentration of AFB1 in the sample, thus realizing the detection of AFB1 in unknown samples.

[0032] Preferably, in step (7), the specific process of obtaining the sample extract is as follows: a certain amount of sample is ground and soaked in a mixture of methanol and ultrapure water, the supernatant is extracted by shaking, and then centrifuged and filtered to obtain the sample extract; the ratio of the amount of sample, methanol and ultrapure water is 5 g: 14 mL: 6 mL; the shaking extraction time is 1 h; the centrifugation speed is 8000 rpm and the time is 15 min; the filter membrane pore size is 0.22 μm.

[0033] The beneficial effects of this invention are:

[0034] (1) The colorimetric-photoenhanced electrochemical dual-mode aptamer sensor of the present invention has a simple construction process, high sensitivity, and good selectivity.

[0035] (2) This invention utilizes photogenerated electrons from a Schottky junction to enhance the sensitivity of the electrochemical aptamer sensor. Compared with other DNA assembly methods, the process is simple and cost-effective. Due to the efficient carrier migration of the constructed rGO-AuNPs Schottky junction, photogenerated electrons can accelerate the electrochemical reduction process of MB, generating an enhanced MB electrochemical signal under ultraviolet light. Therefore, based on photoexcitation signal amplification, higher response sensitivity can be obtained for AFB1 trace analysis, and the sensitivity can be determined by the difference between photoexcitation and photoexcitation. MB The signal undergoes self-testing to improve the reliability and accuracy of the detection.

[0036] (3) The present invention can introduce a portable colorimeter with Bluetooth transmission mode to transmit the solution color to a smartphone, convert it into an RGB digital signal, and realize accurate visualization analysis of AFB1, which is convenient and fast.

[0037] (4) The detection range of AFB1 for the colorimetric mode and electrochemical mode of the dual-mode aptamer sensor constructed in this invention is 5~100 μg mL, respectively. -1 and 1 pg mL -1 ~50 ng / mL -1 The detection limits were 1.23 ng·mL. -1 and 0.12 pg mL -1 Rapid qualitative and precise quantitative analysis of AFB1 enables early warning and accurate monitoring of mold growth. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the construction of a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor for AFB1 detection.

[0039] Figure 2The middle (A) shows the sensor's visual color response to different concentrations of AFB1 (from left to right: 0, 1, 2, 5, 10, 20, 50, 100, 500, 10). 3 5×10 3 , 10 4 5×10 4 , 10 5 ng mL -1 (B) is the calibration curve of ΔC versus the logarithm of AFB1 concentration; (C) is the electrochemical response of the sensor to different concentrations of AFB1 before and after photoexcitation (10). -3 5×10 -3 , 10 -2 5×10 -2 , 10 -1 5×10 -1 , 1, 5, 10, 50 ng mL -1 (D) is I MB Calibration curve with logarithm of AFB1 concentration.

[0040] Figure 3 In the middle, (A) and (D) are the colorimetric and electrochemical signal response diagrams of the sensor to OTA, ZEN, FB1, a mixture of three toxins (OTA+ZEN+FB1), AFB1, and a mixture of four toxins (OTA+ZEN+FB1+AFB1), respectively; (B) and (E) are the reproducibility of seven parallel measurements of colorimetry and electrochemistry, respectively; (C) and (F) characterize the long-term stability of the sensor using colorimetric and electrochemical signal values, respectively. Detailed Implementation

[0041] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments are based on the technical solutions of the present invention and provide detailed implementation steps and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0042] The AFB1 aptamer (Apt) was 5′-Biotin-GTT GGG CAC GTG TTG TCT CTC TGT GTCTCG TGC CCT TCG CTA GGC CCA CA-3′ (50 mer); the primer sequence complementary to the aptamer was 5′-SH-(CH2)6-CAA CTT CTA TGT GGG CCT AGC GAA-3′ (24 mer); and the circular sequence complementary to the primer (Padlock) was 5′-ACA TAG AAG TTG AAG CTG CTA CAA ACG GAG AAA GGA CTC GCA CAACGC AAT CAG GTA TTC GCT AGG CCC-3′ (69 mer). All of these were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0043] Streptavidin magnetic beads (SMBs) were purchased from Beaver Biotechnology Co., Ltd.

[0044] Example 1:

[0045] (1) Preparation of rGO-AuNPs nanomaterials: 2 mL of 1% HAuCl4 solution was added to 2 mL of 2 mg / mL... -1 The rGO dispersion was magnetically stirred for 12 h, followed by centrifugation at 10000 rpm for 15 min. The supernatant was removed, and the precipitate was collected and washed three times with ultrapure water to remove excess HAuCl4, yielding the rGO-AuNPs nanocomposite material. Finally, the obtained rGO-AuNPs nanocomposite material was redispersed in 2 mL of H2O to obtain 2 mg / mL of the product. -1 The rGO-AuNPs nanocomposite dispersion was prepared and stored at 4 °C in the dark for later use.

[0046] (2) Pretreatment of SMBs: Take 100 μL and 10 mg mL of SMBs. -1 Streptavidin magnetic beads (SMBs) were magnetically separated, and the bead protection solution was removed. The precipitate was collected and washed three times with 1 mL Tris-HCl (pH = 7.4) buffer. Finally, the SMBs were magnetically separated again to obtain the SMBs precipitate. The SMBs were redispersed in buffer and resuspended by shaking to obtain an SMBs suspension with a concentration of 2.2 mg / mL. -1 ;

[0047] (3) Preparation of Apt-Primer-MB: 10 μM biotinylated Apt solution and 10 μM Primer solution were mixed in equal volume ratio and heated at 95 °C for 10 min to denature them. Then, the mixture was cooled to room temperature for 20 min to allow the bases to pair, thus obtaining Apt-Primer double-stranded solution. Then, 45 μL of 1 mM MB solution was added to 240 μL of Apt-Primer double-stranded solution and incubated at 37 °C in the dark for 1 h to obtain Apt-Primer-MB solution.

[0048] (4) Preparation of Apt-Primer-MB SMBs: The SMBs suspension prepared in step (2) and the Apt-Primer-MB solution prepared in step (3) were mixed in a ratio of 9:1 and rotated at 30 °C for 75 min to obtain an Apt-Primer-MB SMBs solution. Then, the magnetic beads were magnetically separated and washed three times with Tris-HCl (pH = 7.4) buffer to remove unbound Apt-Primer-MB. Finally, the precipitate obtained after magnetic separation was denoted as Apt-Primer-MB SMBs.

[0049] The application of a colorimetric-photoenhanced electrochemical aptamer sensor based on a bifunctional probe for the detection of aflatoxin B1 follows these steps:

[0050] (1) Pretreatment of GCE: Polish glassy carbon electrode (GCE, Φ = 3 mm) with 0.05 μm aluminum oxide powder until mirror gloss, then sonicate in anhydrous ethanol and ultrapure water for 30 s and then dry in air.

[0051] (2) The 2 mg mL prepared in step (1) of Example 1 -1 The rGO-AuNPs nanocomposite dispersion was used to modify the surface of the pretreated GCE electrode in an amount of 6 μL and dried at room temperature. The modified electrode is denoted as rGO-AuNPs / GCE.

[0052] (3) Mix 100 μL of AFB1 solutions of different concentrations (10 -3 5×10 -3 , 10 -2 5×10 -2 , 10 -1 5×10 -1 ,1, 2, 5, 10, 20, 50, 100, 500, 10 3 5×10 3 , 10 4 5×10 4 , 105 ng mL -1 The Apt-Primer-MB SMBs precipitate prepared in step (4) of Example 1 was added to the precipitate, and the two were in a one-to-one correspondence. Then, the mixture was rotated and mixed at 37 °C for 40 min. After magnetic separation, the supernatant was collected and colorimetrically measured (RGB) using a portable CS-420 spectrophotometer (8 mm aperture, D65 light source, 10° light source angle). The total color difference value ΔC was then calculated according to the Euclidean distance formula. .like Figure 2 As shown in (A), within a certain range, the color of the supernatant gradually deepens with increasing AFB1 concentration. The specific linear relationship is shown in the graph below. Figure 2 As shown in (B), the AFB1 concentration was 5~100 ng / mL -1 and 0.1~100 μg mL -1 Within the range, the logarithm of AFB1 concentration exhibits a good linear relationship with ΔC at both ends, with the linear curves being ΔC = 39.727lgC. AFB1 + 20.617 (R 2 = 0.996) and ΔC = 5.666lgC AFB1 + 70.680 (R 2 = 0.994), proving that the sensor performs well;

[0053] (4) Add 2.5 μM Padlock solution to the supernatant obtained in step (3) and mix at a volume ratio of 1:1. Incubate at 37 °C for 1 h to form a double-chain structure to re-adsorb MB dissociated in the supernatant. Then add 20 μL 1 mM TCEP solution to 200 μL of the mixed solution and react at room temperature for 1 h to activate the thiol group on the Primer chain to obtain Primer-Padlock-MB solution.

[0054] (5) Take the Primer-Padlock-MB solution prepared in step (4) to modify the surface of the rGO-AuNPs / GCE electrode prepared in step (2), the amount is 6 μL, incubate at 4 ℃ for 12 h, and then immerse the modified electrode in 200 μL 5 μMMB solution for 2 min to enhance the MB adsorption. After that, wash the electrode with PBS solution (pH = 7.4), and record it as Primer-Padlock-MB / rGO-AuNPs / GCE;

[0055] (6) Using the modified Primer-Padlock-MB / rGO-AuNPs / GCE from step (5) as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the 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 / cm². 2 Electrochemical signals before and after photoexcitation were recorded using an Autolab PGSTAT302N electrochemical workstation: I MBoff IMB on The test was performed in a 0.1 M PBS buffer solution containing 0.1 M AA at pH 7.4. The scan voltage range was 0 to -0.4 V, the amplitude was 0.025 V, and the frequency was 37 Hz. MBoff and I MBon Two standard curves were constructed by plotting the two signals against the logarithm of AFB1 concentration; as follows: Figure 2 As shown in (C), with the increase of AFB1 concentration, I MBoff and I MBon The sensitivity gradually increases, exhibiting two linear relationships with different linear ranges; the specific linear relationship graph is shown below. Figure 2 As shown in (D), at an AFB1 concentration of 10 pg / mL -1 ~50 ng / mL -1 Within the range, the logarithm of AFB1 concentration and I MBoff It exhibits a good linear relationship, and its linear curve is I. MB(off) =1.987 lgC AFB1 +13.958 (R) 2 = 0.996), at an AFB1 concentration of 1 pg / mL -1 ~5 ng mL -1 Within the range, the logarithm of AFB1 concentration and I MBon It exhibits a good linear relationship, and its linear curve is I. MB(on) =2.795 lgC AFB1 +16.012 (R) 2 = 0.999), proving that the sensor performs well;

[0056] To evaluate the selectivity of the bimodal aptamer sensor, interference experiments were conducted using OTA, ZEN, FB1, a mixture of three toxins (OTA+ZEN+FB1), AFB1, and a mixture of four toxins (OTA+ZEN+FB1+AFB1). Figure 3 As shown in (A) and (D), the signal response of the interfering substance at a concentration 10 times that of AFB1 is negligible compared to the signal intensity generated by AFB1; therefore, the sensor has good selectivity for AFB1 detection.

[0057] Reproducibility and long-term stability are also key factors in evaluating the practicality of a sensor; the reproducibility of the sensor was evaluated by performing seven parallel measurements of AFB1, and the results are as follows: Figure 3 As shown in (B) and (E), △C, I MBoff I MBon The RSDs were 0.7%, 2.8%, and 1.8%, respectively, indicating good reproducibility of the sensor. The sensor was placed in a 4 ℃ dark environment, and the response signal was measured daily to evaluate the long-term stability of the sensor. The results are as follows: Figure 3 As shown in (C) and (F), after the sensor is placed for 7 days, ΔC and I... MBoff I MBon These values ​​represent 97.5%, 97.3%, and 96.7% of the initial signal value, respectively, and exhibit low RSD. These results demonstrate that the dual-mode sensor possesses excellent reproducibility and long-term stability.

[0058] (7) Based on the excellent analytical performance of the dual-mode sensor, actual peanut and peanut soil samples were collected, and AFB1 in the actual samples was analyzed.

[0059] First, obtain the sample extract: take 5 g of peanut sample or peanut soil, grind it, soak it in a mixed solution containing 14 mL of methanol and 6 mL of ultrapure water, shake and extract for 1 h, then centrifuge at 8000 rpm for 15 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the sample extract;

[0060] The corresponding ΔC and I were obtained by colorimetric and electrochemical tests. MBoff I MBon By substituting the numerical value into the constructed standard curve, the concentration of AFB1 in the sample can be determined, thus enabling the detection of AFB1 in unknown samples.

[0061] (a) Take the sample extract and follow the operation of step (3), except that the AFB1 standard solution is replaced with the sample extract; then test the corresponding ΔC value according to the colorimetric mode of step (3); substitute the value into the standard curve constructed in step (3) to obtain the concentration of AFB1 in the sample;

[0062] (b) After replacing the AFB1 standard solution in step (3) with the sample extract to obtain the supernatant, continue with the operations in steps (4) and (5) to obtain the modified electrode after soaking; then test the corresponding I according to the electrochemical mode in step (6). MBoff I MBon Finally, by substituting the numerical value into the standard curve constructed in step (6), the concentration of AFB1 in the sample can be obtained.

[0063] The analytical results are shown in Table 1. Different concentrations of AFB1 in actual peanut samples and peanut soil were analyzed using both colorimetric and electrochemical sensing methods. The recoveries ranged from 92.01% to 101.12% and from 94.82% to 110.29%, respectively. Compared with the national standard method HPLC-FL (90.05% to 108.00%), the proposed dual-mode aptamer sensor device showed higher reliability for the analysis of AFB1 in actual peanut samples and peanut soil.

[0064]

[0065] Note: 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 constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe, 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 rGO-AuNPs nanocomposite material. Finally, the obtained rGO-AuNPs nanocomposite material was redispersed in water to obtain rGO-AuNPs nanocomposite material dispersion. (2) Pretreatment of streptavidin magnetic beads: After magnetic separation, the magnetic bead protection solution was removed from the streptavidin magnetic beads, the precipitate was collected, washed with buffer solution, and then magnetically separated again to obtain SMBs precipitate. Finally, the SMBs precipitate was redispersed in buffer solution and resuspended by shaking to obtain SMBs suspension. (3) Preparation of Apt-Primer-MB: The AFB1 aptamer solution was mixed with the complementary chain solution of AFB1 aptamer and heated to react. After the reaction, it was cooled to room temperature to obtain the Apt-Primer solution. Then, the methylene blue solution was mixed with the Apt-Primer solution and incubated to obtain the Apt-Primer-MB solution. (4) Preparation of Apt-Primer-MB SMBs: Add Apt-Primer-MB solution to the SMBs suspension prepared in step (2), mix by rotation and then perform magnetic separation to obtain the precipitate, which is denoted as Apt-Primer-MB SMBs; (5) Pretreatment of glassy carbon electrode: GCE was polished with aluminum oxide powder, and then ultrasonically treated in anhydrous ethanol and ultrapure water in sequence. After drying, the pretreated GCE electrode was obtained. (6) Modify the rGO-AuNPs nanocomposite dispersion prepared in step (1) onto the surface of the GCE electrode pretreated in step (5), and the electrode after drying is denoted as rGO-AuNPs / GCE; (7) Prepare AFB1 standard solutions of different concentrations and mix them with the prepared Apt-Primer-MB SMBs for incubation. After incubation, collect the supernatant by magnetic separation to obtain supernatant at different concentrations. (8) Take the complementary strand solution of the primer and denot it as Padlock solution; mix Padlock solution with the supernatant prepared in step (7) and incubate. After incubation, add TCEP solution to carry out the activation reaction. After the reaction, Primer-Padlock-MB solution is obtained. (9) Take the Primer-Padlock-MB solution prepared in step (8) and modify it on the surface of rGO-AuNPs / GCE prepared in step (6) above. After incubation, immerse the modified electrode in MB solution. The modified electrode after immersion is called Primer-Padlock-MB / rGO-AuNPs / GCE.

2. The method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe according to claim 1, characterized in that, In step (1), the volume ratio of the chloroauric acid solution to the reduced graphene oxide dispersion is 1:1; wherein the mass concentration of the chloroauric acid solution is 1%, and the concentration of the reduced graphene oxide dispersion is 2 mg / mL. -1 The magnetic stirring time was 12 h; the centrifugation speed was 10000 rpm for 15 min; and the concentration of the rGO-AuNPs nanocomposite dispersion was 2 mg / mL. -1 .

3. The method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe according to claim 1, characterized in that, In step (2), the amount of streptavidin magnetic beads used is 100 μL, and the concentration is 10 mg / mL. -1 The buffer solution is Tris-HCl buffer, pH = 7.4, and the volume of buffer used for each wash is 1 mL; the concentration of the SMBs suspension is 2–3 mg / mL. -1 .

4. The method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe according to claim 1, characterized in that, In step (3), the concentrations of the AFB1 aptamer solution and the complementary chain solution of the AFB1 aptamer are both 10 μM, and the volume ratio is 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 methylene blue solution to the Apt-Primer solution is 45 μL:240 μL, wherein the concentration of the methylene blue solution is 1 mM, the incubation temperature is 37 ℃, and the incubation time is 1 h.

5. The method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe according to claim 1, characterized in that, In step (4), the volume ratio of the SMBs suspension to the Apt-Primer-MB solution is 9:1; the temperature of the mixing reaction is 30 °C and the reaction time is 75 min.

6. The method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe according to claim 1, characterized in that, In step (5), 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; in step (6), the modification amount of the rGO-AuNPs nanocomposite dispersion is 6 μL, and the concentration is 2 mg / mL. -1 .

7. The method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a dual-functional probe according to claim 1, characterized in that, In step (7), the volume ratio of the Apt-Primer-MB SMBs precipitate to the AFB1 standard solution is 1 mg: 100 μL, and the concentration of the AFB1 standard solution is 10. -3 ~10 5 ng mL -1 The incubation temperature is 37 °C and the incubation time is 40 min; in step (8), the volume ratio of the supernatant, Padlock solution and TCEP solution is 5:5:1, wherein the concentration of Padlock is 2.5 μM and the concentration of TCEP solution is 1 mM; the incubation temperature is 37 °C and the time is 1 h; the activation reaction temperature is room temperature and the reaction time is 1 h.

8. The method for constructing a colorimetric-photoenhanced electrochemical dual-mode aptamer sensor based on a bifunctional probe according to claim 1, characterized in that, In step (9), the amount of Primer-Padlock-MB solution used for modification is 6 μL, the incubation temperature is 4 ℃, and the incubation time is 12 h; the amount of MB solution used is 200 μL, the concentration is 5 μM, and the soaking time is 2 min.

9. The use of the colorimetric-photoenhanced electrochemical dual-mode aptamer sensor device based on a bifunctional probe prepared according to any one of claims 1-8 for the detection of AFB1, characterized in that, The steps are as follows: Construction of the S1 standard curve: (a) Colorimetric mode: Using the supernatant prepared in step (7) of claim 1 as the test object, the RGB values ​​are obtained and recorded. The total color difference value ΔC is calculated according to the Euclidean distance formula. A standard curve is constructed by using the colorimetric signal ΔC and the logarithm of the corresponding AFB1 concentration. (b) Electrochemical mode: Using the Primer-Padlock-MB / rGO-AuNPs / GCE modified in step (9) of claim 1 as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, electrochemical detection was performed; the working interface was irradiated with an external light source, and the electrochemical signals before and after photoexcitation were obtained and recorded as I MBoff and I MBon ; with I MBoff and I MBon Two standard curves were constructed by comparing the two signals with the logarithm of AFB1 concentration. Detection of AFB1 in actual S2 samples: (a) First, obtain the sample extract, replace the AFB1 standard solution with the sample extract, and then operate according to the colorimetric mode in step S1 (a) to test and obtain the corresponding ΔC value; substitute the value into the standard curve constructed in step S1 (a) to obtain the concentration of AFB1 in the sample, and realize the detection of AFB1 in unknown samples. (b) After replacing the AFB1 standard solution with the sample extract to obtain the supernatant, the modified electrode after soaking is obtained according to steps (8) and (9) of claim 1; then the electrochemical mode of step S1 (b) is operated to test and obtain the corresponding I MBoff I MBon The value is then substituted into the standard curve constructed in step S1(b) to obtain the concentration of AFB1 in the sample, thus realizing the detection of AFB1 in unknown samples.

10. The use according to claim 9, characterized in that, In step S1(a), the colorimetric determination is performed by recording and detecting RGB values ​​using a CS-420 spectrophotometer with an aperture of 8 mm, a light source type of D65, and a light source angle of 10°. In step S1(b), the electrochemical detection was recorded using an Autolab PGSTAT 302N electrochemical workstation. The scanning voltage range was 0 to -0.4 V, the amplitude was 0.025 V, and the frequency was 37 Hz. The light source wavelength was 365 nm, and the power was 7 W / cm². 2 The vertical distance between the light source and the working interface was 2 cm; the test solution was 0.1 M PBS buffer with pH=7.4, containing ascorbic acid at a final concentration of 0.1 M.

11. The use according to claim 9, characterized in that, In step S2, the specific process for obtaining the sample extract is as follows: a certain amount of sample is ground and then soaked in a mixture of methanol and ultrapure water. The supernatant is extracted by shaking, followed by centrifugation and filtration to obtain the sample extract. The ratio of sample, methanol, and ultrapure water is 5 g: 14 mL: 6 mL. The shaking extraction time is 1 h. The centrifugation speed is 8000 rpm and the time is 15 min. The filter membrane pore size is 0.22 μm.

Images