CN113237868B

CN113237868B - Ratio type detection method of surface enhanced Raman sensor based on graphene oxide to mycotoxin

Info

Publication number: CN113237868B
Application number: CN202110730935.5A
Authority: CN
Inventors: 马小媛; 陈沛芳; 李晨彪; 王周平
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-06-29
Filing date: 2021-06-29
Publication date: 2022-04-05
Anticipated expiration: 2041-06-29
Also published as: CN113237868A

Abstract

The invention provides a ratio type detection method of a graphene oxide-based surface-enhanced Raman sensor on mycotoxin, and belongs to the technical field of spectral analysis. The invention constructs a ratio type detection method of a graphene oxide-based surface enhanced Raman sensor to mycotoxin, synthesizes a high-performance probe (Au-4MBA @ AgNPs-ToxinAPt) and a high-stability substrate (ITO/Au/GO), then prepares a sensor (Au-4MBA @ AgNPs-ToxinAPt-GO/Au/ITO), and realizes successful construction by utilizing pi-pi interaction of an aptamer and the surface of graphene oxide. The method realizes the qualitative and quantitative detection of high sensitivity and high selectivity of mycotoxin, and is suitable for the fields of food safety and analysis and detection.

Description

Ratio type detection method of surface enhanced Raman sensor based on graphene oxide to mycotoxin

Technical Field

The invention belongs to the technical field of spectral analysis, and particularly relates to a ratio type detection method of a graphene oxide-based surface-enhanced Raman sensor on mycotoxin.

Background

Mycotoxins are metabolites of fungi produced under suitable conditions during harvest, processing, storage and transport of agricultural products, such as Aflatoxins (AFs), ochratoxin a (ota), Zearalenone (ZEN) and Patulin (PAT). The aflatoxins are a group of harmful toxins produced by aspergillus flavus and aspergillus parasiticus, and mainly comprise AFB1, AFB2, AFG1, AFG2, AFM1 and AFM 2. Aflatoxin B1 is classified as group 1 carcinogen by International cancer research organization (2002), its acute toxicity is 10 times that of potassium cyanide and 68 times that of arsenic, and its chronic toxicity is liable to induce canceration and mutation. In daily life, agricultural products such as peanuts, corns and grains are easy to be polluted. But due to their chemical structure stability, they are generally difficult to destroy at processing temperatures, and therefore pose a serious threat to human health. The national quality control bureau stipulates that the quality control bureau is one of the necessary items.

At present, the traditional instrumental analysis methods are widely applied, mainly including High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), liquid chromatography-mass spectrometry (LC-MS) and Thin Layer Chromatography (TLC), and although these analysis techniques can realize sensitive and reliable detection, the operation is complex, the detection speed is slow, the cost is high, and the sample pretreatment is complicated. The immunoassay method is a series of immunoassay methods derived by mainly utilizing the specific recognition effect of antigens and antibodies, and commonly used enzyme-linked immunosorbent assay (ELISA), immunofluorescence labeling technology, immunoaffinity column method (IAC), immune colloidal gold technology (ICS) and the like, and can achieve high-precision and high-specificity detection, but false positive results are easy to occur in toxin detection, and the detection range needs to be improved. In addition, researchers have studied a variety of fingerprint spectroscopy techniques to analyze mycotoxins, such as near infrared spectroscopy (NIRS), mid infrared spectroscopy (MIRS), and High Spectral Imaging (HSI). Near infrared spectroscopy is a technique in which different chemical bonds in a sample to be measured are affected by changes in vibrational energy during irradiation. NIRS and MIRS both have the advantages of high detection efficiency and high signal-to-noise ratio, but have low specificity and lack of characteristic signal peaks. While HSI is an emerging technology that combines spectroscopic and imaging methods. It provides spectral and spatial information of the analyte, but the detection efficiency and specificity are low. Therefore, all these fingerprints have a low sensitivity in the detection of mycotoxins. Secondly, electrochemical, fluorescent and colorimetric sensors have the advantage of rapid detection, but false negative results are easy to occur.

Surface Enhanced Raman Spectroscopy (SERS) refers to a technique in which a laser is irradiated onto a rough substrate surface to cause a chemical or physical change, resulting in a significant enhancement of the raman signal. The accepted enhancement mechanism is mainly due to electromagnetic enhancement caused by the optical properties of the nanomatrix (EF: 10)⁶-10⁸) And chemical enhancement due to charge transfer between the substrate and the sample (EF: 10²). Among other things, electromagnetic field enhancement is a major contribution to SERS signal enhancement. SERS has the excellent characteristics of high sensitivity, high specificity, simple pretreatment, nondestructive testing and the like. The research on domestic and foreign documents shows that: firstly, the preparation of a high-performance SERS probe in the sensor plays an important role in the detection result, but some Raman signal molecules marked on the surface of a noble metal are easily interfered by external conditions, so that the SERS signal is unstable. Secondly, the SERS substrate of the nano hybrid film type has good SERS enhancement effect, but the lack of internal standard signal limits the application of the SERS substrate in quantitative analysis. Meanwhile, the existing SERS technology is still less in ratio type detection method for mycotoxin, and the stability and reproducibility of SERS signals are poor. Therefore, there remains a challenge in developing high performance enhanced substrates and probes. In order to rapidly monitor and control the contamination of mycotoxinsThe research focuses on developing simple, efficient and high-sensitivity SERS aptamer sensors.

Disclosure of Invention

In order to solve the technical problems, the invention provides a ratio type detection method of a graphene oxide-based surface-enhanced Raman sensor on mycotoxin. The preparation method has the advantages of simple process, short time, low cost and the like in the preparation process, and can be applied to large-scale production.

A graphene oxide-based surface enhanced Raman sensor ratiometric detection method of mycotoxins, comprising the steps of:

(1) preparing Au-4MBA @ Ag NPs-ToxinAPt:

s1: phosphorylating Au-4MBA @ Ag NPs to obtain phosphorylated Au-4MBA @ Ag NPs; the phosphorylated coating can improve the stability of the nanoparticles in a high salt environment;

s2: activating a disulfide bond in the sulfhydrylation mycotoxin aptamer to obtain an activated sulfhydrylation mycotoxin aptamer SH-ToxinAPt;

s3: mixing and incubating the phosphorylated Au-4MBA @ Ag NPs obtained in S1 and the activated thiol mycotoxin aptamer obtained in S2 to obtain Au-4MBA @ Ag NPs-ToxinAPt; dropwise adding a NaCl aqueous solution in the mixed incubation process, wherein the concentration of the NaCl aqueous solution is 0.5-2 mol/L;

(2) preparing an ITO/Au/GO substrate:

performing electrodeposition on ITO glass in a chloroauric acid aqueous solution with the mass fraction of 0.5-4% to obtain a gold film, and dripping an aqueous solution of a PBS buffer solution of graphene oxide on the surface of the gold film to obtain the ITO/Au/GO substrate; the solution is quickly and uniformly spread due to the good hydrophilicity of the gold film; adopting a potentiostatic method for electrodeposition, wherein the electrodes are as follows: ITO glass, Ag/AgCl and platinum wire were used as working electrode, reference electrode and counter electrode, respectively. The chemical enhancement effect of the graphene oxide is utilized to improve the signal intensity of the probe sensor.

(3) Constructing an Au-4MBA @ AgNPs-ToxinApt-GO/Au/ITO sensor and detecting:

step I: immersing the ITO/Au/GO substrate obtained in the step (2) into the Au-4MBA @ Ag NPs-ToxinAPt obtained in the step (1) for incubation reaction to obtain the Au-4MBA @ AgNPs-ToxinAPt-GO/Au/ITO sensor; wherein the other end of ToxinApt is adsorbed on the surface of the graphene oxide through pi-pi interaction.

Step II: and (3) mixing and incubating the Au-4MBA @ AgNPs-ToxinAPt-GO/Au/ITO sensor obtained in the step (I) with a mycotoxin solution, detecting the ratio Raman signal intensity of the sensor, and performing qualitative or quantitative analysis.

In one embodiment of the invention, the concentration of the mycotoxin solution is in the range of 0.0001-100 ng/mL.

In one embodiment of the present invention, in step (1) S1, the Au-4MBA @ Ag NPs are prepared by: and adding 20-40mM sodium citrate solution into the Au-4MBA NPs solution, uniformly mixing, sequentially adding 8-12mM silver nitrate solution and 8-12mM ascorbic acid solution respectively, and performing incubation reaction to obtain the Au-4MBA @ Ag NPs. Wherein, the sodium citrate solution is mixed evenly to protect the nano particles.

In one embodiment of the present invention, in step (1), in S1, the phosphorylation method of Au-4MBA @ Ag NPs is: and mixing and incubating Au-4MBA @ Ag NPs and bis (p-sulfonylphenyl) phenylphosphine dihydrate dipotassium salt (BSPP) to obtain the phosphorylated Au-4MBA @ Ag NPs.

In one embodiment of the present invention, in step (1), in S2, the method for activating the disulfide bond in the thiolated mycotoxin aptamer comprises: mixing the aqueous solution of Tris-HCl buffer solution of the thiolated mycotoxin aptamer with trichloroethyl phosphate (TCEP) for activation reaction to obtain the activated thiolated mycotoxin aptamer.

In one embodiment of the invention, the molar concentration ratio of the Tris-HCl buffer solution of the thiolated mycotoxin aptamer to trichloroethyl phosphate (TCEP) is 1: 50-200.

In one embodiment of the present invention, in S3 of step (1), the molar concentration ratio of the phosphorylated Au-4MBA @ Ag NPs to the activated mercaptomycotoxin aptamer is 1:1 to 10.

In one embodiment of the present invention, in the step (2), the concentration of the aqueous solution of the graphene oxide in the PBS buffer solution is 0.02-0.08 mg/mL.

In one embodiment of the present invention, in step (3), the standard curve for the quantitative analysis is prepared as follows: soaking the Au-4MBA @ AgNPs-ToxinApt-GO/Au/ITO sensor in mycotoxin solutions with different concentrations, standing and incubating for 0.5-4h, cleaning and drying the sensor, separating some probes from the surface of graphene oxide due to high affinity between an aptamer and a target, detecting a Raman spectrogram under 633nm laser excitation, and taking 1078cm in the obtained Raman spectrogram^-1Intensity of Raman signal I₁₀₇₄And 1330cm^-1Intensity of Raman signal I₁₃₃₀The ratio of (d) is the ordinate and the logarithm of the concentration of the mycotoxin solution is taken as the abscissa to obtain a standard curve.

Meanwhile, after the substrate and the probe are compounded, the Raman signal is enhanced again and can be attributed to the combined effects of the following two resonances: plasma effect of AuNPs (electromagnetic field enhancement) and GO-induced charge transfer effect (chemical enhancement).

In one embodiment of the invention, the mycotoxins include aflatoxin, ochratoxin a, zearalenone, and patulin.

Thiol-modified AFB1 aptamer chain: 5 '-SH-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CC-3'.

(1) The invention respectively prepares a high-performance probe (Au-4MBA @ Ag NPs-ToxinAPt) and a high-stability substrate (ITO/Au/GO), realizes the successful construction of a sensor (Au-4MBA @ AgNPs-ToxinAPt-GO/Au/ITO) by utilizing the pi-pi interaction between an aptamer and the surface of graphene oxide, and designs a high-sensitivity ratio type aptamer sensor for the detection of mycotoxin by combining the specific recognition effect of the appropriate ligand on a target object.

(2) According to the invention, Raman signal molecules are selected to be embedded into the Au @ Ag core-shell nano particles, signals are not easily influenced by the surrounding complex environment, excellent stability is shown, and meanwhile, the composite Raman active material integrates the advantages of various nano materials, so that the probe has more stable and stronger Raman enhancement effect, and is used as a probe for developing a high-performance sensor.

(3) The enhanced substrate can be used for preparing the gold film by combining a rapid electrodeposition method and preparing the graphene oxide film by a simple dropping coating method only in a short time (less than 30 minutes), and meanwhile, the good hydrophilicity of the gold film enables the graphene oxide solution to be quickly and uniformly spread.

(4) Compared with the conventional Raman signal molecule as an internal standard, the graphene oxide-based inert two-dimensional nanomaterial can provide more active sites for connecting nucleic acid chains while correcting result errors, and further improve the stability of the substrate in a complex environment.

(5) Compared with the method for constructing the sensor by utilizing the principle of complementary pairing of two nucleic acid chain bases, the method has the advantages that the sensor Au-4MBA @ Ag NPs-ToxinAPt-GO/Au/ITO can be assembled only by relying on pi-pi interaction between the graphene oxide and the aptamer chain, and the strategy plays roles in saving cost and simplifying operation to a certain extent.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the ratio type detection method for mycotoxin by using the graphene oxide-based surface enhanced Raman sensor comprises the steps of synthesizing a high-performance probe (Au-4MBA @ Ag NPs-ToxinAPt) and a high-stability substrate (ITO/Au/GO), successfully constructing by using an aptamer and a pi-pi interaction sensor (Au-4MBA @ AgNPs-ToxinAPt-GO/Au/ITO) on the surface of graphene oxide, and characterizing and selecting a D-band peak of the graphene oxide as an internal standard peak (IS) and 1078cm of 4-mercaptobenzoic acid by using a Raman spectrometer^-1As a signal peak. The detection system has maximum adsorption amount of substrate surface to probe in absence of mycotoxin, I₁₀₇₈/I₁₃₃₀To a maximum value; in the presence of mycotoxin, partial Raman probes are separated from the surface of the substrate due to the high affinity of the mycotoxin aptamer and the mycotoxin, so that the I of the 4-mercaptobenzoic acid₁₀₇₈Decrease so that I is within a certain range₁₀₇₈/I₁₃₃₀Decreasing with increasing mycotoxin concentration, appears to be inversely related. The ratio type detection method can greatly reduce the influence of poor Raman signal reproducibility caused by the change of external environmental factors, thereby improving the reliability of the result. The method is suitable for the food safety field and the analysis and detection field.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a transmission electron microscopy characterization (TEM) of Au NPs (A), Au-4MBA @ Ag NPs (B) obtained in example 1 of the present invention; UV-visible spectra (C) of Au NPs, Au-4MBA NPs, Au-4MBA @ Ag NPs; surface enhanced Raman spectroscopy (D) of Au NPs, Au-4MBA NPs, Au-4MBA @ Ag NPs.

FIG. 2 is a Scanning Electron Microscope (SEM) representation of ITO/Au (A) and ITO/Au/GO (B) obtained in example 1 of the present invention; a surface enhanced Raman spectrum (C) of ITO, ITO/Au, ITO/Au/GO; ITO/Au, ITO/Au/GO soaked in 1mM 4-MBA solution and then surface enhanced Raman spectrum (D).

FIG. 3 is a scanning electron microscope characterization (SEM) (A) of Au-4MBA @ AgNPs-AFB1Apt-GO/Au/ITO obtained in example 1 of the present invention; ITO/Au/GO, Au-4MBA @ AgNPs-AFB1Apt and Au-4MBA @ AgNPs-AFB1Apt-GO/Au/ITO surface enhanced Raman spectra (B).

FIG. 4 is a Raman spectrum (A) of aflatoxin at various concentrations in example 1 of the present invention; corresponding standard curve (B).

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

Step 1) preparation of Au-4MBA @ Ag NPs:

au NPs: all used glass containers and rotors are soaked in aqua regia overnight before preparation, and are cleaned and dried by ultrapure water for reuse. 99mL of ultrapure water and 1mL of 4% chloroauric acid solution were added to a 250mL round-bottom flask and heated with stirring until the solution boiled, 15mL of 1% sodium citrate solution was added rapidly, the solution changed color from colorless, black, dark purple to wine-red within one minute, the heating was continued at reflux for 20min to allow complete reduction of the nanoparticles, and finally the heat source was turned off and cooled to room temperature with stirring. The characterization result of the Au NPs is shown in figure 1.

Au-4MBA NPs: 2mL of the AuNPs sol prepared above was centrifuged once at 9000rpm for 30min, and the precipitate was redissolved in 0.5mL of ultrapure water. Then 5. mu.L of 1mM 4-mercaptobenzoic acid (4-MBA) ethanol solution was added to the above solution, the final concentration was maintained at 10. mu.M, and after overnight shaking incubation at room temperature, the solution was centrifuged at 10000rpm for 10min to remove excess signal molecules, and then re-dissolved in the same volume of ultrapure water.

Au-4MBA @ Ag NPs: adding 80 mu L of 38.8mM sodium citrate solution into the solution, uniformly mixing to protect nano particles, adding 120 mu L of 10mM silver nitrate solution under the condition of oscillation, then slowly dropwise adding 200 mu L of 10mM ascorbic acid solution, oscillating and incubating for 30min to reduce silver nitrate to form a silver shell with a certain thickness outside a gold core, finally centrifuging for 10min at 10000rpm to remove redundant reaction solvent, then re-dissolving in 2mL of ultrapure water, and storing in a refrigerator at 4 ℃ for later use. The characterization results of the Au-4MBA @ Ag NPs are shown in figure 1.

Step 2) preparing Au-4MBA @ Ag NPs-AFB1 Apt:

phosphorylated Au-4MBA @ Ag NPs: mixing a certain volume of Au-4MBA @ Ag NPs prepared in the step 1) with bis (p-sulfonylphenyl) phenylphosphine dihydrate dipotassium salt (BSPP) to keep the final concentration of the Au-4MBA @ Ag NPs to be 0.1mg/mL, improving the stability of the nanoparticles in a high-salt environment by virtue of a phosphorylation coating, oscillating and incubating for 8 hours at room temperature, centrifuging for 20min at 10000rpm, redissolving and concentrating for four times for later use.

Activated SH-AFB1 Apt: AFB1Apt is dissolved in a certain Tris-HCl buffer solution (Tris-HCl buffer solution: 25mM Tris-HCl, 140mM NaCl and 5mM KCl) to prepare a solution with the concentration of 100 mu M, and then the solution is mixed with 10mM trichloroethyl phosphate (TCEP) solution in equal volume for activating sulfydryl for 1h, so that the disulfide bond in the sulfydryl aptamer can be reduced.

Au-4MBA @ Ag NPs-AFB1 Apt: the phosphorylated Au-4MBA @ Ag NPs and the activated SH-AFB1Apt are incubated at room temperature in a shaking way, and NaCl solution is gradually dripped in the other 12 hours to enable the final concentration to reach 0.1M so as to increase the combination amount of the two. Finally, the mixture was centrifuged at 9000rpm for 20min to remove excess free nucleic acids, and redissolved in Tris-HCl buffer and stored in a refrigerator at 4 ℃ until use.

Thiol-modified AFB1 aptamer chain: 5 '-SH-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCTTCG CTAGGC CC-3'.

Step 3) preparing ITO/Au/GO:

ITO: the ITO conductive glass was cut into small pieces of 1X 2 cm. Before the preparation of the SERS substrate, the ITO glass is subjected to ultrasonic treatment in acetone, ethanol and ultrapure water for 10min respectively and then dried by nitrogen for later use.

ITO/Au: the conductive surface of the glass is determined by a multimeter, 1 × 1cm bare ITO glass is immersed in a 1% tetrachloroauric acid solution, a dense gold film is formed by electrodeposition for 200s by a potentiostatic method at a potential of-0.2V (three electrodes: ITO glass, Ag/AgCl and platinum wire are respectively used as a working electrode, a reference electrode and a counter electrode), after electrodeposition is completed, the substrate is immersed in ultrapure water twice to remove residues, and the substrate is dried at 37 ℃ for standby.

ITO/Au/GO: dissolving 1mg of graphene oxide powder in 1mL of 1 XPBS buffer solution, continuously performing ultrasonic treatment for 6h at the water temperature of 18 ℃ and the power of 40% to form a stable and clear brown yellow solution, preparing a solution of 1mg/mL, and diluting by 32 times for later use. And then uniformly dripping 30 mu L of graphene oxide solution on the surface of the gold film, rapidly and uniformly spreading the solution due to good hydrophilicity of the gold film, and drying the solution at 37 ℃ for later use, wherein the characterization results of the ITO/Au and the ITO/Au/GO are shown in figure 2.

Step 4) constructing an Au-4MBA @ AgNPs-AFB1Apt-GO/Au/ITO sensor: immersing the substrate prepared in the step 3) into the SERS probe solution prepared in the step 2) for standing incubation for 3h at room temperature, and adsorbing the other end of the AFB1Apt on the surface of graphene oxide through pi-pi interaction, thereby forming the Au-4MBA @ AgNPs-AFB1Apt-GO/Au/ITO sensor. After the incubation is finished, the surface of the ITO is slowly washed by Tris-HCl buffer solution to remove redundant probes on the surface, and finally dried at 37 ℃ for later use. The characterization result of Au-4MBA @ AgNPs-AFB1Apt-GO/Au/ITO is shown in figure 3.

Step 5) establishing a standard curve for detecting AFB 1: soaking the sensor constructed in the step 4) in a certain amount of AFB1 (0, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100ng/mL) with different concentrations, standing and incubating for 2h at room temperature, separating some probes from the surface of the graphene oxide due to high affinity between the aptamer and the target, washing the substrate with Tris-HCl solution for 2 times, and then drying in the air under appropriate conditions. Collecting Raman spectrum on the substrate under 633nm laser excitation by Raman spectrometer, using logarithmic value of AFB1 concentration as abscissa, 4-mercaptobenzoic acid at 1078cm^-1And graphene oxide at 1330cm^-1Ratio-type Raman intensity (I) of (A)₁₀₇₄/I₁₃₃₀) As an ordinate, a standard curve was determined, and the experimental results are shown in FIG. 4. The experimental results show that: log₁₀C_AFB1And I₁₀₇₈/I₁₃₃₀Presents a good linear relation, and the corresponding regression equation is 1.07-0.44X (correlation coefficient R)²0.99), the constructed SERS aptamer sensor can realize ratio-type detection on AFB1, and the D band peak (1330 cm) of graphene oxide is illustrated^-1) Can be used as an ideal reference signal value to improve the quantitative detection capability of the sensor on AFB 1.

Example 2

Step 1) preparation of Au-4MBA @ Ag NPs:

au NPs: all used glass containers and rotors are soaked in aqua regia overnight before preparation, and are cleaned and dried by ultrapure water for reuse. 99mL of ultrapure water and 1mL of 4% chloroauric acid solution were added to a 250mL round-bottom flask and heated with stirring until the solution boiled, 15mL of 1% sodium citrate solution was added rapidly, the solution changed color from colorless, black, dark purple to wine-red within one minute, the heating was continued at reflux for 20min to allow complete reduction of the nanoparticles, and finally the heat source was turned off and cooled to room temperature with stirring.

Au-4MBA @ Ag NPs: adding 80 mu L of 38.8mM sodium citrate solution into the solution, uniformly mixing to protect nano particles, adding 150 mu L of 8mM silver nitrate solution under the condition of oscillation, then slowly dropwise adding 167 mu L of 12mM ascorbic acid solution, oscillating and incubating for 30min to reduce silver nitrate to form a silver shell with a certain thickness outside a gold core, finally centrifuging for 10min at 10000rpm to remove redundant reaction solvent, then re-dissolving in 2mL of ultrapure water, and storing in a refrigerator at 4 ℃ for later use.

Step 2) preparing Au-4MBA @ Ag NPs-OTAApt:

Activated SH-OTAApt: OTAApt is dissolved in a certain Tris-HCl buffer solution (Tris-HCl buffer solution: 25mM Tris-HCl, 140mM NaCl and 5mM KCl) to prepare a solution with the concentration of 100 mu M, and then the solution is mixed with 5mM trichloroethyl phosphate (TCEP) solution in equal volume for activating sulfydryl for 1h, so that disulfide bonds in the sulfydryl aptamer can be reduced.

Au-4MBA @ Ag NPs-OTAApt: the phosphorylated Au-4MBA @ Ag NPs and the activated SH-AFB1Apt are incubated at room temperature in a shaking way, and NaCl solution is gradually dripped in the other 12 hours to enable the final concentration to reach 0.1M so as to increase the combination amount of the two. Finally, the mixture was centrifuged at 9000rpm for 20min to remove excess free nucleic acids, and redissolved in Tris-HCl buffer and stored in a refrigerator at 4 ℃ until use.

Thiol-modified OTA aptamer chain: 5 '-SH-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGGACA-3'.

Step 3) preparing ITO/Au/GO:

ITO/Au/GO: dissolving 1mg of graphene oxide powder in 1mL of 1 XPBS buffer solution, continuously performing ultrasonic treatment for 6h at the water temperature of 18 ℃ and the power of 40% to form a stable and clear brown yellow solution, preparing a solution of 1mg/mL, and diluting by 32 times for later use. And then uniformly dripping 30 mu L of graphene oxide solution on the surface of the gold film, quickly and uniformly spreading the solution due to good hydrophilicity of the gold film, and drying at 37 ℃ for later use.

Step 4) constructing an Au-4MBA @ AgNPs-OTAApt-GO/Au/ITO sensor: immersing the substrate prepared in the step 3) into the SERS probe solution prepared in the step 2) for standing incubation for 3 hours at room temperature, and adsorbing the other end of the OTAApt on the surface of the graphene oxide through pi-pi interaction, thereby forming the Au-4MBA @ AgNPs-OTAApt-GO/Au/ITO sensor. After the incubation is finished, the surface of the ITO is slowly washed by Tris-HCl buffer solution to remove redundant probes on the surface, and finally dried at 37 ℃ for later use.

Step 5) establishing a standard curve for detecting OTA: and (3) soaking the sensor constructed in the step 4) in OTA with a certain amount of different concentrations (0, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100ng/mL), standing and incubating for 0.8h at room temperature, separating some probes from the surface of the graphene oxide due to high affinity between the aptamer and the target, washing the substrate with Tris-HCl solution for 2 times, and then drying in the air under a proper condition. Collecting Raman spectrum on the substrate under 633nm laser excitation by Raman spectrometer, using log value of OTA concentration as abscissa, 4-mercaptobenzoic acid at 1078cm^-1And graphene oxide at 1330cm^-1Ratio-type Raman intensity (I) of (A)₁₀₇₄/I₁₃₃₀) As an ordinate, a standard curve was determined. Log₁₀C_OTAAnd I₁₀₇₈/I₁₃₃₀Presents a good linear relation, verifies that the constructed SERS aptamer sensor can realize the ratio type detection of OTA, and simultaneously shows the D band peak (1330 cm) of graphene oxide^-1) Can be used as an ideal reference signal value to improve the quantitative detection capability of the sensor on OTA.

Example 3

Step 1) preparation of Au-4MBA @ Ag NPs:

Au-4MBA @ Ag NPs: adding 80 mu L of 38.8mM sodium citrate solution into the solution, uniformly mixing to protect nano particles, adding 100 mu L of 12mM silver nitrate solution under the condition of oscillation, then slowly dropwise adding 250 mu L of 8mM ascorbic acid solution, oscillating and incubating for 30min to reduce silver nitrate to form a silver shell with a certain thickness outside a gold core, finally centrifuging for 10min at 10000rpm to remove redundant reaction solvent, then re-dissolving in 2mL of ultrapure water, and storing in a refrigerator at 4 ℃ for later use.

Step 2) preparing Au-4MBA @ Ag NPs-AFB1 Apt:

Activated SH-AFB1 Apt: AFB1Apt is dissolved in a certain Tris-HCl buffer solution (Tris-HCl buffer solution: 25mM Tris-HCl, 140mM NaCl and 5mM KCl) to prepare a solution with the concentration of 100 mu M, and then the solution is mixed with 20mM trichloroethyl phosphate (TCEP) solution in equal volume for activating sulfydryl for 1h, so that the disulfide bond in the sulfydryl aptamer can be reduced.

Thiol-modified AFB1 aptamer chain: 5 '-SH-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTAGGC CC-3'.

Step 3) preparing ITO/Au/GO:

ITO/Au/GO: dissolving 1mg of graphene oxide powder in 1mL of 1 XPBS buffer solution, continuously performing ultrasonic treatment for 6h at the water temperature of 18 ℃ and the power of 40% to form a stable and clear brown yellow solution, preparing a solution of 1mg/mL, and diluting the solution to 0.06mg/mL for later use. And then uniformly dripping 30 mu L of graphene oxide solution on the surface of the gold film, quickly and uniformly spreading the solution due to good hydrophilicity of the gold film, and drying at 37 ℃ for later use.

Step 4) constructing an Au-4MBA @ AgNPs-AFB1Apt-GO/Au/ITO sensor: immersing the substrate prepared in the step 3) into the SERS probe solution prepared in the step 2) for standing incubation for 3h at room temperature, and adsorbing the other end of the AFB1Apt on the surface of graphene oxide through pi-pi interaction, thereby forming the Au-4MBA @ AgNPs-AFB1Apt-GO/Au/ITO sensor. After the incubation is finished, the surface of the ITO is slowly washed by Tris-HCl buffer solution to remove redundant probes on the surface, and finally dried at 37 ℃ for later use.

Step 5) establishing a standard curve for detecting AFB 1: soaking the sensor constructed in the step 4) in a certain amount of AFB1 (0, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100ng/mL) with different concentrations, standing and incubating for 1.5h at room temperature, separating some probes from the surface of the graphene oxide due to high affinity between the aptamer and the target, washing the substrate with a Tris-HCl solution for 2 times, and then drying in the air under appropriate conditions. Collecting Raman spectrum on the substrate under 633nm laser excitation by Raman spectrometer, using logarithmic value of AFB1 concentration as abscissa, 4-mercaptobenzoic acid at 1078cm^-1And graphene oxide at 1330cm^-1Ratio-type Raman intensity (I) of (A)₁₀₇₄/I₁₃₃₀) As an ordinate, a standard curve was determined.

Example 4

Step 1) preparation of Au-4MBA @ Ag NPs:

au NPs: all used glass containers and rotors are soaked in aqua regia overnight before preparation, and are cleaned and dried by ultrapure water for reuse. 99mL of ultrapure water and 1mL of 4% chloroauric acid solution were added to a 250mL round-bottom flask and heated with stirring until the solution boiled, 15mL of 1% sodium citrate solution was added quickly, the solution changed color from colorless, black, dark purple to wine-red within one minute, the condensing was continued under heating with reflux for 20min to allow complete reduction of the nanoparticles, and finally the heat source was turned off and cooled to room temperature with stirring.

Au-4MBA @ Ag NPs: adding 80 mu L of 38.8mM sodium citrate solution into the solution, uniformly mixing to protect nano particles, adding 150 mu L of 8mM silver nitrate solution under the condition of oscillation, then slowly dropwise adding 200 mu L of 10mM ascorbic acid solution, oscillating and incubating for 30min to reduce silver nitrate to form a silver shell with a certain thickness outside a gold core, finally centrifuging for 10min at 10000rpm to remove redundant reaction solvent, then re-dissolving in 2mL of ultrapure water, and storing in a refrigerator at 4 ℃ for later use.

Step 2) preparing Au-4MBA @ Ag NPs-AFB1 Apt:

Activated SH-AFB1 Apt: AFB1Apt is dissolved in a certain Tris-HCl buffer solution (Tris-HCl buffer solution: 25mM Tris-HCl, 140mM NaCl and 5mM KCl) to prepare a solution with the concentration of 100 mu M, and then the solution is mixed with 5mM trichloroethyl phosphate (TCEP) solution in equal volume for activating sulfydryl for 1h, so that the disulfide bond in the sulfydryl aptamer can be reduced.

Step 3) preparing ITO/Au/GO:

ITO/Au/GO: dissolving 1mg of graphene oxide powder in 1mL of 1 XPBS buffer solution, continuously performing ultrasonic treatment for 6h at the water temperature of 18 ℃ and the power of 40% to form a stable and clear brown yellow solution, preparing a solution of 1mg/mL, and diluting the solution to 0.04mg/mL for later use. And then uniformly dripping 30 mu L of graphene oxide solution on the surface of the gold film, quickly and uniformly spreading the solution due to good hydrophilicity of the gold film, and drying at 37 ℃ for later use.

Step 5) establishing a standard curve for detecting AFB 1: soaking the sensor constructed in the step 4) in a certain amount of AFB1 (0, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100ng/mL) with different concentrations, standing and incubating for 1h at room temperature, separating some probes from the surface of the graphene oxide due to high affinity between the aptamer and the target, washing the substrate with Tris-HCl solution for 2 times, and then drying in the air under appropriate conditions. Collecting Raman spectrum on the substrate under 633nm laser excitation by Raman spectrometer, using logarithmic value of AFB1 concentration as abscissa, 4-mercaptobenzoic acid at 1078cm^-1And graphene oxide at 1330cm^-1Ratio-type Raman intensity (I) of (A)₁₀₇₄/I₁₃₃₀) As an ordinate, a standard curve was determined.

Example 5

Step 1) preparation of Au-4MBA @ Ag NPs:

Au-4MBA @ Ag NPs: adding 80 mu L of 38.8mM sodium citrate solution into the solution, uniformly mixing to protect nano particles, adding 167 mu L of 8mM silver nitrate solution under the condition of oscillation, then slowly dropwise adding 250 mu L of 8mM ascorbic acid solution, oscillating and incubating for 30min to reduce silver nitrate to form a silver shell with a certain thickness outside a gold core, finally centrifuging for 10min at 10000rpm to remove redundant reaction solvent, then re-dissolving in 2mL of ultrapure water, and storing in a refrigerator at 4 ℃ for later use.

Step 2) preparing Au-4MBA @ Ag NPs-AFB1 Apt:

Activated SH-AFB1 Apt: AFB1Apt is dissolved in a certain Tris-HCl buffer solution (Tris-HCl buffer solution: 25mM Tris-HCl, 140mM NaCl and 5mM KCl) to prepare a solution with the concentration of 100 mu M, and then the solution is mixed with 12mM trichloroethyl phosphate (TCEP) solution in equal volume for activating sulfydryl for 1h, so that the disulfide bond in the sulfydryl aptamer can be reduced.

Step 3) preparing ITO/Au/GO:

ITO/Au/GO: dissolving 1mg of graphene oxide powder in 1mL of 1 XPBS buffer solution, continuously performing ultrasonic treatment for 6h at the water temperature of 18 ℃ and the power of 40% to form a stable and clear brown yellow solution, preparing a solution of 1mg/mL, and diluting the solution to 0.05mg/mL for later use. And then uniformly dripping 30 mu L of graphene oxide solution on the surface of the gold film, quickly and uniformly spreading the solution due to good hydrophilicity of the gold film, and drying at 37 ℃ for later use.

Step 5) establishing a standard curve for detecting AFB 1: soaking the sensor constructed in the step 4) in a certain amount of AFB1 (0, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100ng/mL) with different concentrations, standing and incubating for 0.5h at room temperature, separating some probes from the surface of the graphene oxide due to high affinity between the aptamer and the target, washing the substrate with a Tris-HCl solution for 2 times, and then drying in the air under appropriate conditions. Collecting Raman spectrum on the substrate under 633nm laser excitation by Raman spectrometer, using logarithmic value of AFB1 concentration as abscissa, 4-mercaptobenzoic acid at 1078cm^-1And graphene oxide at 1330cm^-1Ratio-type Raman intensity (I) of (A)₁₀₇₄/I₁₃₃₀) As an ordinate, a standard curve was determined.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A graphene oxide-based surface-enhanced Raman sensor ratio-based detection method for mycotoxin, which is characterized by comprising the following steps:

(1) preparing Au-4MBA @ Ag NPs-ToxinAPt:

s1: phosphorylating Au-4MBA @ Ag NPs to obtain phosphorylated Au-4MBA @ Ag NPs; the phosphorylation method of the Au-4MBA @ Ag NPs comprises the following steps: mixing and incubating Au-4MBA @ Ag NPs and dihydrate bis (p-sulfonylphenyl) phenylphosphine dipotassium salt to obtain the phosphorylated Au-4MBA @ Ag NPs, and keeping the final concentration of the Au-4MBA @ Ag NPs at 0.1 mg/mL;

s2: activating a disulfide bond in the thiolated mycotoxin aptamer to obtain an activated thiolated mycotoxin aptamer;

(2) preparing an ITO/Au/GO substrate:

performing electrodeposition on ITO glass in a chloroauric acid aqueous solution to obtain a gold film, and dripping an aqueous solution of a PBS buffer solution of graphene oxide on the surface of the gold film to obtain the ITO/Au/GO substrate;

(3) constructing an Au-4MBA @ AgNPs-ToxinApt-GO/Au/ITO sensor and detecting:

step I: immersing the ITO/Au/GO substrate obtained in the step (2) into the Au-4MBA @ Ag NPs-ToxinAPt obtained in the step (1) for incubation reaction to obtain the Au-4MBA @ AgNPs-ToxinAPt-GO/Au/ITO sensor;

2. The ratio-based assay of claim 1, wherein in step (1) S1, the Au-4MBA @ Ag NPs are prepared by: and adding 20-40mM sodium citrate solution into the Au-4MBA NPs solution, uniformly mixing, respectively adding 8-12mM silver nitrate solution and 8-12mM ascorbic acid solution, and performing incubation reaction to obtain the Au-4MBA @ Ag NPs.

3. The ratio-based assay of claim 1, wherein in step (1) S2, the method for activating the disulfide bond in the thiolated mycotoxin aptamer comprises: and mixing the aqueous solution of the Tris-HCl buffer solution of the thiolated mycotoxin aptamer with trichloroethyl phosphate for activation reaction to obtain the activated thiolated mycotoxin aptamer.

4. The ratio-based assay of claim 3, wherein the molar ratio of aqueous Tris-HCl buffer to trichloroethyl phosphate of the thiolated mycotoxin aptamer is between 1:50 and 200.

5. The ratio-based assay of claim 1, wherein in step (1) S3, the ratio of the amount of phosphorylated Au-4MBA @ Ag NPs to the amount of activated mercaptomycotoxin aptamer is from 1:1 to 10.

6. The ratio-based assay of claim 1, wherein in step (2), the concentration of the graphene oxide in PBS buffer is 0.02-0.08 mg/mL.

7. The ratio assay of claim 1, wherein in step (3), the standard curve for the quantitative assay is prepared as follows: soaking the Au-4MBA @ AgNPs-ToxinApt-GO/Au/ITO sensor in mycotoxin solutions with different concentrations, standing and incubating for 0.5-4h, cleaning and drying the sensor, detecting a Raman spectrogram under laser excitation, and taking 1078cm in the obtained Raman spectrogram^-1Intensity of Raman signal I₁₀₇₈And 1330cm^-1Intensity of Raman signal I₁₃₃₀The ratio of (d) is the ordinate and the logarithm of the concentration of the mycotoxin solution is taken as the abscissa to obtain a standard curve.

8. The ratio assay of claim 7, wherein the concentration of said mycotoxin solution is in the range of 0.0001-100 ng/mL.

9. The ratio assay of claim 8, wherein the mycotoxin is aflatoxin, ochratoxin a, zearalenone, or patulin.