CN-116773461-B - Construction method and application of colorimetric-optical enhancement electrochemical dual-mode aptamer sensing device based on dual-function probe

CN116773461BCN 116773461 BCN116773461 BCN 116773461BCN-116773461-B

Abstract

The invention belongs to the technical field of biosensors, and particularly relates to a construction method and application of a colorimetric-optical enhancement electrochemical dual-mode aptamer sensor based on a dual-function probe. The invention obtains a double signal channel of probe Methylene Blue (MB) by utilizing streptavidin magnetic beads, obtains supernatant with color discrimination through the specific release of MB at a magnetic bead interface for colorimetric analysis, researches an electron transfer path of a reduced graphene oxide-gold nano ion Schottky junction, promotes the oxidation-reduction process of MB through photo-generated electrons, thereby realizing electrochemical signal amplification, can obtain higher response sensitivity based on light excitation signal amplification so as to perform AFB1 trace analysis, and can perform self-detection through I MB signals before and after light excitation so as to improve the reliability and accuracy of detection, and can realize early warning and accurate monitoring of agricultural sample mildew analysis by rapid qualitative and accurate quantification of the AFB 1.

Inventors

LIU DONG
WEI YA
LI YUYE
LIU SHUDA
MENG SHUYUN
YOU TIANYAN

Assignees

江苏大学

Dates

Publication Date: 20260512
Application Date: 20230629

Claims (11)

1. The construction method of the colorimetric-optical enhancement electrochemical dual-mode aptamer sensing device based on the dual-function probe is characterized by comprising the following steps: (1) Adding chloroauric acid solution into reduced graphene oxide dispersion liquid, magnetically stirring, centrifuging to remove supernatant, collecting precipitate, washing with ultrapure water, centrifuging to obtain rGO-AuNPs nano composite material, and finally re-dispersing the obtained rGO-AuNPs nano composite material in water to obtain rGO-AuNPs nano composite material dispersion liquid; (2) The pretreatment of streptavidin magnetic beads comprises removing magnetic bead protecting solution after magnetic separation of streptavidin magnetic beads, collecting precipitate, adding buffer solution for washing, performing magnetic separation to obtain SMBs precipitate, and finally re-dispersing SMBs precipitate in the buffer solution for shaking and re-suspending to obtain SMBs suspension; (3) The preparation of the Apt-Primer-MB comprises the steps of mixing an AFB1 aptamer solution with a complementary strand solution of the AFB1 aptamer, heating for reaction, cooling to room temperature after the reaction to obtain an Apt-Primer solution, mixing a methylene blue solution with the Apt-Primer solution, and incubating to obtain the Apt-Primer-MB solution; (4) Apt-Primer-MB SMBs, namely adding the Apt-Primer-MB solution into the SMBs suspension prepared in the step (2), performing magnetic separation after rotating and mixing reaction to obtain a precipitate which is named as Apt-Primer-MB SMBs; (5) The pretreatment of the glassy carbon electrode, namely polishing and grinding the GCE by using aluminum oxide powder, sequentially carrying out ultrasonic treatment in absolute ethyl alcohol and ultrapure water, and drying to obtain a pretreated GCE electrode; (6) Modifying the rGO-AuNPs nanocomposite dispersion liquid prepared in the step (1) to the surface of the GCE electrode pretreated in the step (5), and marking the electrode after drying as rGO-AuNPs/GCE; (7) Preparing AFB1 standard solutions with different concentrations, respectively mixing and incubating with the prepared Apt-Primer-MB SMBs, and collecting supernatant after incubation through magnetic separation to obtain supernatant corresponding to different concentrations; (8) Mixing and incubating the Padlock solution with the supernatant prepared in the step (7), adding a TCEP solution for activation reaction after incubation, and obtaining a Primer-Padlock-MB solution after reaction; (9) And (3) modifying the surface of the rGO-AuNPs/GCE prepared in the step (6) by using the Primer-Padlock-MB solution prepared in the step (8), incubating, and soaking the modified electrode in the MB solution after incubation, wherein the soaked modified electrode is marked as the Primer-Padlock-MB/rGO-AuNPs/GCE.
2. The method for constructing a dual-functional probe-based colorimetric-photo-enhanced electrochemical dual-mode aptamer sensor device is characterized in that in the step (1), the volume ratio of chloroauric acid solution to reduced graphene oxide dispersion liquid is 1:1, the mass concentration of the chloroauric acid solution is 1%, the concentration of the reduced graphene oxide dispersion liquid is 2mg mL -1 , the magnetic stirring time is 12 h, the centrifugal speed is 10000 rpm, the time is 15 min, and the concentration of the rGO-AuNPs nanocomposite dispersion liquid is 2mg mL -1 .
3. The method for constructing the colorimetric-photo-enhanced electrochemical dual-mode aptamer sensor based on the bifunctional probe of claim 1, wherein in the step (2), the dosage of streptavidin magnetic beads is 100 μl, the concentration is 10mg mL -1 , the buffer solution is Tris-HCl buffer solution, the pH=7.4, the dosage of each washing buffer solution is 1 mL, and the concentration of the SMBs suspension is 2-3 mg mL -1 .
4. The method for constructing a dual-functional probe-based colorimetric-photoenhanced electrochemical dual-mode aptamer sensor according to claim 1, wherein in the step (3), the concentration of the AFB1 aptamer solution and the concentration of the complementary strand solution of the AFB1 aptamer are 10 μm, the volume ratio is 1:1, the temperature of the heating reaction is 95 ℃, the time is 10 min, the cooling time is 10-20 min, the usage ratio of the methylene blue solution to the Apt-Primer solution is 45 μl to 240 μl, the concentration of the methylene blue solution is 1mM, the incubation temperature is 37 ℃, and the incubation time is 1 h.
5. The method for constructing a dual-functional probe-based colorimetric-photo-enhanced electrochemical dual-mode aptamer sensor device according to claim 1, wherein in the step (4), the volume ratio of SMBs suspension to Apt-Primer-MB solution is 9:1, the temperature of the mixing reaction is 30 ℃, and the reaction time is 75 min.
6. The method for constructing a dual-functional probe-based colorimetric-photoenhanced electrochemical dual-mode aptamer sensor according to claim 1, wherein in the step (5), the diameter of the GCE electrode is 3 mm, the particle size of the aluminum oxide powder used is 0.05 μm, the ultrasonic treatment time is 30s, and in the 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 dual-functional probe-based colorimetric-photopheresis electrochemical dual-mode aptamer sensor according to claim 1, wherein in the step (7), the dosage ratio of the Apt-Primer-MB SMBs precipitate to the AFB1 standard solution is 1 mg/100 μl, the concentration of the AFB1 standard solution is 10 -3 ~10 5 ng mL -1 , the incubation temperature is 37 ℃ and the incubation time is 40min, the volume ratio of the supernatant, the Padlock solution and the TCEP solution is 5:5:1, the concentration of the Padlock is 2.5 μΜ, the concentration of the TCEP solution is 1mM, the incubation temperature is 37 ℃, the time is 1h, the temperature of the activation reaction is room temperature, and the reaction time is 1 h.
8. The method for constructing a dual-functional probe-based colorimetric-photo-enhanced electrochemical dual-mode aptamer sensor device according to claim 1, wherein in the step (9), the modification amount of the Primer-pad-MB solution is 6 μl, the incubation temperature is 4 ℃, the incubation time is 12 h, the use amount of the MB solution is 200 μl, the concentration is 5 μΜ, and the soaking time is 2 min.
9. Use of a dual function probe based colorimetric-photoenhanced electrochemical dual mode aptamer sensor device prepared according to any of claims 1-8 for detecting AFB1, characterized by the steps of: Construction of an S1 standard curve: (a) A colorimetric mode, wherein the supernatant prepared in the step (7) of claim 1 is taken as a test object, RGB values are obtained by obtaining records, a total color difference value delta C is obtained by calculation according to an Euclidean distance formula, and a standard curve is constructed by using the logarithm of the colorimetric signal delta C and the corresponding AFB1 concentration; (b) An electrochemical mode, namely, using a Primer-Padlock-MB/rGO-AuNPs/GCE modified in the step (9) of the claim 1 as a working electrode, using a saturated Ag/AgCl electrode as a reference electrode and a platinum wire electrode as a counter electrode for electrochemical detection, irradiating a working interface by using an external light source, and obtaining electrochemical signals before and after recording light excitation as I MBoff and I MBon respectively, and respectively constructing two standard curves by using logarithms of the I MBoff and I MBon signals and the AFB1 concentration; S2 detection of AFB1 in actual sample: (a) Firstly, obtaining a sample extracting solution, replacing an AFB1 standard solution with the sample extracting solution, and then operating according to a colorimetric mode in the step S1, and testing to obtain a corresponding delta C value, wherein the value is substituted into a standard curve constructed in the step S1, so that the concentration of the AFB1 in the sample can be obtained, and the detection of the AFB1 in an unknown sample can be realized; (b) And (3) after the AFB1 standard solution is replaced by the sample extracting solution to obtain a supernatant, the soaked modified electrode is obtained according to the operations of the steps (8) and (9) of the claim 1, then the electrochemical mode operation is carried out according to the step (b) of the step S1, the corresponding I MBoff 、I MBon value is obtained through testing, and finally the value is substituted into the standard curve constructed in the step (b) of the step S1, so that the concentration of the AFB1 in the sample can be obtained, and the detection of the AFB1 in the unknown sample can be realized.
10. The use according to claim 9, wherein in step S1 (a), the colorimetric measurement is performed by recording the detected RGB values with a CS-420 spectrocolorimeter, the caliber is 8mm, the light source type is D65, and the light source angle is 10 °; In step S1 (b), the electrochemical detection was recorded and obtained by an electrochemical workstation of model Autolab PGSTAT 302N, the scanning voltage range was 0 to-0.4V, the amplitude was 0.025V, the frequency was 37 Hz, the light source wavelength was 365 nm, the power was 7W/cm 2 , the vertical distance of the light source from the working interface was 2 cm, the test solution was 0.1M, and the PBS buffer with pH=7.4 contained ascorbic acid at a final concentration of 0.1M.
11. The method according to claim 9, wherein in the step S2, the specific process of obtaining the sample extracting solution is that a certain amount of sample is ground and then is soaked in a mixed solution of methanol and ultrapure water, the supernatant is extracted by shaking, and then the supernatant is centrifuged and filtered to obtain the sample extracting solution, wherein the dosage of the sample, the methanol and the ultrapure water is 5 g:14 mL:6 mL, the shaking extraction time is 1h, the rotational speed of the centrifugation is 8000 rpm, the time is 15min, and the pore diameter of the filter membrane is 0.22 μm.

Description

Construction method and application of colorimetric-optical enhancement electrochemical dual-mode aptamer sensing device based on dual-function probe Technical Field The invention belongs to the technical field of biosensors, and particularly relates to a construction method and application of a colorimetric-optical enhancement electrochemical dual-mode aptamer sensing device based on a dual-function probe. Background Aflatoxin B1 (AFB 1) is a secondary metabolite of aspergillus flavus and aspergillus parasiticus, and is a common pathogenic bacterium in agricultural products. Has high toxicity and carcinogenicity, can cause acute hepatitis and hemorrhagic necrosis, and can have adverse effects on organism growth and immune disease resistance even if exposed at low level. Therefore, it is required to develop a sensor excellent in performance to realize rapid and accurate detection of AFB 1. Electrochemical sensors have received great attention because of their rapid response, high sensitivity and specificity. However, the practical application of electrochemical sensors in AFB1 mildew monitoring remains challenging (1) unavoidable matrix interference may lead to reduced electrochemical signal of probe molecules, limiting ultrasensitive detection of AFB1 trace contamination in complex sample matrices. The method has the advantages that (1) compared with visual analysis of a colorimetric sensor, the application of the electrochemical sensor in field detection is limited by a relatively complex reading mode of the electrochemical sensor, and the design of a dual-mode sensing method ensures that a sensing platform not only maintains the respective distinct advantages of each mode, but also mutually compensates the defects, and can effectively improve the analysis performance. The introduction of multiple indication probes can effectively complete the construction of a dual-mode sensing platform, but relatively complicated assembly, such as deposition, etching and enzyme-catalyzed chromogenic substrate treatment, limit the practical application to a certain extent, so that development of a simple and efficient multifunctional probe indication strategy with dual-signal response is highly needed. Disclosure of Invention The invention aims to invent a colorimetric-photo-enhanced electrochemical dual-mode aptamer sensing device based on a difunctional probe Methylene Blue (MB) by utilizing photo-generated electron transfer of a Schottky junction, and the rapid and accurate detection of AFB1 is realized. The invention utilizes streptavidin magnetic beads to obtain a double-signal channel of a probe MB, and obtains supernatant with color identification degree for colorimetric analysis through the specific release of MB at the interface of the magnetic beads. And the electron transfer path of the reduced graphene oxide-gold nanoparticle (rGO-AuNPs) Schottky junction is studied, and the oxidation-reduction process of MB is promoted by photo-generated electrons, so that the electrochemical signal amplification is realized. In addition, the dual-mode aptamer sensor developed by the invention can carry out high-sensitivity and high-selectivity detection on AFB1 in peanut and peanut soil samples, and provides a powerful tool for mildew analysis of agricultural samples. The aim of the invention is achieved by the following technical scheme: The invention firstly provides a preparation method of a colorimetric-optical enhancement electrochemical dual-mode aptamer sensing device, which comprises the following steps: (1) The rGO-AuNPs nano material is prepared by adding chloroauric acid (HAuCl 4) solution into reduced graphene oxide (rGO) dispersion, magnetically stirring, centrifuging to remove supernatant, collecting precipitate, washing with ultrapure water, centrifuging to obtain rGO-AuNPs nano composite material, finally, redispersing the obtained rGO-AuNPs nano composite material in water to obtain rGO-AuNPs nano composite material dispersion, wherein in the step (1), the volume ratio of HAuCl 4 solution to rGO dispersion is 1:1, the mass concentration of HAuCl 4 solution is 1%, the concentration of rGO dispersion is 2 mg mL -1, the magnetic stirring time is 12 h, the centrifugal speed is 10000 rpm, the time is 15 min, and the concentration of rGO-AuNPs nano composite material dispersion is 2 mg mL -1. (2) The pretreatment of Streptavidin Magnetic Beads (SMBs) comprises the steps of removing a magnetic bead protection solution after magnetic separation of the streptavidin magnetic beads, collecting a precipitate, adding a buffer solution for washing, then carrying out magnetic separation to obtain a SMBs precipitate, finally dispersing the SMBs precipitate in the buffer solution again, and shaking and re-suspending to obtain SMBs suspension, wherein in the step (2), the dosage of the streptavidin magnetic beads is 100 mu L, the concentration is 10 mg mL -1, the buffer solution is Tris-HCl buffer solution, the pH=7.4, the d