CN114034681A - Surface-enhanced Raman spectroscopy analysis and detection method of zearalenone and application thereof - Google Patents
Surface-enhanced Raman spectroscopy analysis and detection method of zearalenone and application thereof Download PDFInfo
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- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 title claims abstract description 89
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 title claims abstract description 87
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 238000004458 analytical method Methods 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 21
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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Abstract
The invention discloses a surface-enhanced Raman spectroscopy analysis and detection method of zearalenone and application thereof. The surface enhanced Raman spectroscopy analysis and detection method of zearalenone comprises the following steps: s100, preparing a standard sample: mixing the zearalenone standard solutions with different concentrations with the premixed solution; s200, taking porous carbon nitride modified by hydrophobic dendritic gold-copper composite nanoparticles as an SERS substrate, obtaining the concentration relation between the peak value of the characteristic Raman displacement position of 3,3 ', 5, 5' -tetramethyl benzidine and zearalenone, and establishing a standard curve; s300, detecting the concentration of the zearalenone in the sample to be detected, and calculating the concentration of the zearalenone in the mixed solution to be detected according to the standard curve in the step S200. The method has high selectivity, sensitivity, rapidity and accuracy. The method can be used for detecting zearalenone.
Description
Technical Field
The invention belongs to the field of chemical analysis and detection, and particularly relates to a surface-enhanced Raman spectroscopy analysis and detection method of zearalenone and application thereof.
Background
Zearalenone, also known as F-2 toxin, is a common mycotoxin widely found in various crops, such as corn, wheat, barley, oats, and other grains, and in food products produced from such grains, such as corn oil, soy sauce, vinegar, and the like. The toxicity of zearalenone is mainly manifested by affecting the reproductive system of human and animals and higher carcinogenicity and teratogenicity. Therefore, how to judge the zearalenone content in the food has important significance for guaranteeing the food safety. In addition, some illegal merchants adopt zearalenone-polluted food raw materials for production and processing in order to improve profits, which greatly increases the occurrence of zearalenone poisoning events. Therefore, the establishment of a rapid, accurate, stable and sensitive method for analyzing zearalenone in food is of great significance.
The prior detection methods of zearalenone which are widely applied comprise thin-layer chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay and the like, but the thin-layer chromatography is poor in quantitative analysis, the enzyme-linked immunosorbent assay is poor in specificity, the high performance liquid chromatography needs to be combined with fluorescence detection, the operation is complex, and the analysis time is long.
Disclosure of Invention
The present invention is directed to solving at least one of the above problems in the prior art. Therefore, the invention provides a surface enhanced Raman spectroscopy analysis and detection method of zearalenone.
The second aspect of the invention provides an application of the surface enhanced Raman spectroscopy detection method of zearalenone to the concentration of zearalenone in corn.
The first aspect of the invention provides a surface enhanced Raman spectroscopy analysis and detection method of zearalenone, which comprises the following steps:
s100, preparing a standard sample: mixing gold nanoparticles, zearalenone aptamer, 3 ', 5, 5' -tetramethylbenzidine and hydrogen peroxide to obtain a premixed solution; mixing the zearalenone standard solutions with different concentrations with the premixed solution to react to obtain zearalenone mixed solutions with different standard concentrations;
s200, taking porous carbon nitride modified by hydrophobic dendritic gold-copper composite nanoparticles as an SERS substrate, obtaining the concentration relation between the peak value of the characteristic Raman displacement position of 3,3 ', 5, 5' -tetramethyl benzidine and zearalenone, and establishing a standard curve;
s300, detecting the concentration of the zearalenone in a sample to be detected: and (3) mixing the sample to be detected with the premixed solution obtained in the step (S100) for reaction to obtain a mixed solution to be detected, detecting the peak value of the characteristic Raman shift of the 3,3 ', 5, 5' -tetramethylbenzidine according to the detection method same as that in the step (S200), and calculating the concentration of the zearalenone in the mixed solution to be detected according to the standard curve in the step (S200).
The principle of the invention is as follows: the nano gold has better catalytic performance, so that the 3,3 ', 5, 5' -tetramethyl benzidine can be catalyzed into the oxidized 3,3 ', 5, 5' -tetramethyl benzidine in the presence of hydrogen peroxide, and the SERS signal is weakened. The aptamer is wrapped on the surface of the nanogold through electrostatic adsorption, so that the catalytic action of the aptamer can be inhibited. When zearalenone exists, the aptamer is subjected to morphological change and is separated from the nanogold due to a strong recognition effect between the aptamer and the zearalenone, and the nanogold can have a catalytic effect to catalyze the 3,3 ', 5, 5' -tetramethylbenzidine into the oxidized 3,3 ', 5, 5' -tetramethylbenzidine. Therefore, the higher the zearalenone concentration is, the more the detached aptamers are, the better the catalytic performance of the nanogold is, and the more the oxidized 3,3 ', 5, 5' -tetramethyl benzidine is, the weaker the SERS signal is; conversely, the stronger the SERS signal.
The technical scheme of the surface enhanced Raman spectroscopy analysis method of zearalenone of the invention at least has the following beneficial effects:
according to the method, the porous carbon nitride modified by the hydrophobic dendritic gold-copper composite nanoparticles is used as an SERS substrate, and the concentration of the zearalenone is determined through the recognition effect between the zearalenone and a zearalenone aptamer.
According to some embodiments of the present invention, the sample to be tested is further pre-processed in step S300.
According to some embodiments of the invention, the zearalenone aptamer has the sequence 5'-GATGGGGAAAGGGTCCCCCTGGGTTGGAGCATCGGACA-3'.
According to some embodiments of the invention, the gold nanoparticles have an average particle size of 30 to 70 nm.
According to some embodiments of the invention, the zearalenone aptamer has a concentration of 10 to 1000 nmol/L.
According to some embodiments of the invention, the concentration of the 3,3 ', 5, 5' -tetramethylbenzidine is 1 to 20 mg/L.
According to some embodiments of the invention, the concentration of the hydrogen peroxide is 1 to 500 μ M.
According to some embodiments of the present invention, in step S200, the hydrophobic dendritic gold-copper composite nanoparticle modified porous carbon nitride substrate is prepared by the following method:
s210, adding a gold source, a copper source and a reducing agent into a porous carbon nitride solution, carrying out solvothermal reaction, and adding a hydrophobic reagent for continuous reaction;
s220, filtering the product in the step S210, and distributing the filtered solid in a solid support to obtain a hydrophobic solid SERS substrate; the solid support is chromatographic paper and/or filter paper.
According to some embodiments of the invention, the concentration of the porous carbon nitride solution in step S210 is 1.42-4.27 mg/mL.
According to some embodiments of the present invention, the solvothermal reaction temperature in step S210 is 95-105 ℃.
According to some embodiments of the invention, the gold source is from chloroauric acid.
According to some embodiments of the invention, the copper source is from copper chloride and/or copper nitrate.
According to the second aspect of the invention, the application of the surface enhanced Raman spectroscopy method for zearalenone in detection of zearalenone is provided.
Drawings
FIG. 1 is a graph showing SERS response of a zearalenone molecule quantitatively analyzed by example 1 of the present invention;
FIG. 2 is a graph showing the working curve of SERS quantitative detection of zearalenone by example 1 of the present invention;
fig. 3 is a bar graph of SERS response of different zearalenone analogs and zearalenone.
Detailed Description
The following are specific examples of the present invention, and the technical solutions of the present invention will be further described with reference to the examples, but the present invention is not limited to the examples.
Example 1
s210, adding 7.5mL of 10.0mmol/L chloroauric acid, 7.5mL of 10.0mmol/L copper chloride and 0.7mL of 1.0mol/L glucose solution into 2.84mg/mL of porous carbon nitride solution, carrying out solvothermal reaction, heating to 100 ℃, and preserving heat for 30 min; adding 75.0mg of hexadecylamine to continue the reaction;
s220, filtering the product obtained in the step S210, and distributing the filtered solid in a solid support to obtain the hydrophobic dendritic gold-copper composite nanoparticle modified porous carbon nitride SERS substrate.
A surface enhanced Raman spectroscopy analysis and detection method of zearalenone comprises the following steps:
s100, preparing a standard sample: adding 50 mu L of 100nmol/L zearalenone aptamer into 50 mu L of nano gold solution, then respectively adding 50 mu L of 10 mu M hydrogen peroxide and 100 mu L of 6.0 mg/L3, 3 ', 5, 5' -tetramethyl benzidine, and fully mixing to obtain a premixed solution; mixing 50 mu L of zearalenone with mass concentrations of 0.1 mu g/L, 0.2 mu g/L, 0.5 mu g/L, 1.0 mu g/L, 2.0 mu g/L, 5.0 mu g/L, 10.0 mu g/L, 20.0 mu g/L, 50.0 mu g/L, 100.0 mu g/L, 200.0 mu g/L and 500.0 mu g/L with the premixed solution, and reacting for 15min to obtain zearalenone mixed solutions with different standard concentrations;
s200, establishing a standard curve: dropwise adding the zearalenone mixture with different standard concentrations into the prepared SERS substrate of the porous carbon nitride modified by the hydrophobic dendritic gold-copper composite nanoparticles, and mixing the zearalenone mixtures with different concentrationsAnd (3) directly detecting by using a Delta Nu Raman instrument, wherein the excitation intensity is 48mV, the integration time is 5s, each concentration is continuously tested for 3 times, and the 3 data are used for statistics to obtain an average value and a relative deviation. 1330cm of 3,3 ', 5, 5' -tetramethylbenzidine-1The peak intensity of the characteristic peak was used for quantification. Establishing a standard curve according to the concentration relation between the peak value of the characteristic Raman shift and the zearalenone;
pretreating a sample to be detected: 40.0g of crushed corn and oat samples are respectively put into a homogenizing cup, 4g of sodium chloride and 100mL of 90% acetonitrile solution (v/v) are added, the mixture is stirred and extracted for 2min at a high speed by a homogenizer, and the mixture is quantitatively filtered by filter paper. And (3) transferring 10.0mL of filtrate, adding 40mL of water for dilution and mixing uniformly, and filtering by using glass fiber filter paper until the filtrate is clear for later use. The immunoaffinity column was attached under a glass syringe, and 10.0mL of the filtrate was accurately removed and injected into the glass syringe. The air pressure pump is connected with the glass syringe, and the pressure is adjusted to ensure that the solution slowly passes through the immunoaffinity column at the flow rate of 1-2 drops/s until part of air enters the affinity column. The column was rinsed 1 time with 5mL of water at a flow rate of 1-2 drops/s until a portion of the air entered the affinity column, and all the effluent was discarded. Accurately add 1.5mL of methanol to elute at a flow rate of about 1 drop/s. Collecting eluate in glass test tube, blowing to dryness at 55 deg.C under nitrogen, dissolving residue with 1.0mL ultrapure water, filtering with 0.22 μm microporous membrane, and collecting filtrate.
S300, detecting the concentration of the zearalenone in a sample to be detected: respectively mixing the treated 50 mu L of sample to be detected with the premixed liquid obtained in the step S100, reacting for 15min to obtain a mixed liquid to be detected, detecting the peak value of the characteristic Raman shift of 3,3 ', 5, 5' -tetramethylbenzidine according to the same detection method as the step S200, and calculating 1330cm of 3 data-1And substituting the average value and the relative standard deviation of the peak values into a zearalenone concentration standard curve to obtain the corn, wherein the zearalenone concentration in the oat sample is respectively 4.6 (+ -0.17) mu g/kg and 8.8 (+ -0.55) mu g/kg.
In order to verify the accuracy of the zearalenone detection method of example 1 in detecting zearalenone, the solution to be detected in example 4 is subjected to a labeling treatment, a labeling sample is prepared by adding 1.0 μ g/kg, 20.0 μ g/kg and 200.0 μ g/kg zearalenone standard solutions to the sample to be detected, then performing SERS detection, continuously testing for 3 times, calculating the average value and the relative deviation of peaks at 1330cm-1 of 3 data, substituting the average value and the relative deviation into a zearalenone concentration standard curve, so as to obtain the zearalenone concentration in the labeling sample, and calculating the labeling recovery rate of the sample to be 97.5-102.9% and the relative standard deviation to be 3.4-8.2%.
The detection accuracy of the established SERS analysis method is verified by high performance liquid chromatography-mass spectrometry (HPLC-MS). The treated sample liquid to be tested was subjected to HPLC-MS measurement using a high performance liquid chromatography-mass spectrometer supplied by Shimadzu corporation, Japan, and the column used was a C18 column (50 mm. times.2.0 mm, 2 μm). Acetonitrile-water is selected as a mobile phase, gradient elution is adopted, and the elution conditions are as follows: 0-5 min: mobile phase a rose from 25% to 70%; 5-6 min: mobile phase a was maintained at 70%; 6-9 min: mobile phase a decreased from 70% to 25%. The flow rate was 0.2mL/min and the amount of sample was 5. mu.L. The mass spectrum conditions are as follows: ionization mode, electrospray ionization (ESI-); capillary voltage is 3.0 kV; the source temperature is 120 ℃; the temperature of the desolventizing agent is 350 ℃; taper hole airflow: nitrogen at the flow rate of 100L/h; desolventizing the gas stream: nitrogen at the flow rate of 600L/h; collision gas: argon gas, the collision pressure is 2.60 multiplied by 10 < -4 > Pa; the scanning mode is as follows: scanning negative ions; parent ion (m/z): 317.1; ionic ion (m/z): 174.9/273.9. The corn is obtained through HPLC-MS detection, the concentrations of zearalenone in the oat sample are respectively 4.4 (+ -0.17) mu g/kg and 9.1 (+ -0.38) mu g/kg, and the relative deviation with the SERS analysis method is less than 5.2%, so that the reliability of the analysis method is proved.
Example 2
This example 2 was identical to example 1 in raw materials and testing methods, except that the test sample sources were two different corn oils and 3 data were calculated at 1330cm-1The mean value and the relative standard deviation of the peak values are substituted into a zearalenone concentration standard curve to obtain the zearalenone concentrations of 137.0 (+ -4.80) mu g/kg and 115.6 (+ -6.01) mu g/kg in 2 corn oil samples respectively.
To verify the accuracy of the SERS detection method of zearalenone in example 5 in detecting zearalenone, the solution to be detected in example 4 was subjected to labeling treatmentThe standard sample is prepared by adding 1.0 μ g/kg, 20.0 μ g/kg and 200.0 μ g/kg zearalenone standard solution to the sample to be tested, performing SERS detection, continuously testing for 3 times, and calculating 1330cm of 3 data-1And substituting the average value and the relative deviation of the peak values into a zearalenone concentration standard curve to obtain the zearalenone concentration in the standard sample, and calculating to obtain the standard sample recovery rate of 102.3-108.8% and the relative standard deviation of 3.2-7.3%.
The detection accuracy of the established SERS analysis method is verified by high performance liquid chromatography-mass spectrometry (HPLC-MS). The treated sample liquid to be tested was subjected to HPLC-MS measurement using a high performance liquid chromatography-mass spectrometer supplied by Shimadzu corporation, Japan, and the column used was a C18 column (50 mm. times.2.0 mm, 2 μm). Acetonitrile-water is selected as a mobile phase, gradient elution is adopted, and the elution conditions are as follows: 0-5 min: mobile phase a rose from 25% to 70%; 5-6 min: mobile phase a was maintained at 70%; 6-9 min: mobile phase a decreased from 70% to 25%. The flow rate was 0.2mL/min and the amount of sample was 5. mu.L. The mass spectrum conditions are as follows: ionization mode, electrospray ionization (ESI-); capillary voltage is 3.0 kV; source temperature: 120 ℃; the temperature of the desolventizing agent is 350 ℃; taper hole airflow: nitrogen at the flow rate of 100L/h; desolventizing the gas stream: nitrogen at the flow rate of 600L/h; collision gas: argon gas, the collision pressure is 2.60 multiplied by 10 < -4 > Pa; the scanning mode is as follows: scanning negative ions; parent ion (m/z): 317.1; ionic ion (m/z): 174.9/273.9. The corn is obtained through HPLC-MS detection, the concentrations of zearalenone in the oat sample are 135.1 (+ -0.54) mu g/kg and 113.5 (+ -0.79) mu g/kg respectively, and the relative deviation with the SERS analysis method is less than 1.8%, so that the method is proved to have good accuracy.
FIG. 1 shows 1330cm of zearalenone and 3,3 ', 5, 5' -tetramethylbenzidine at different concentrations in example 1 of the present invention-1The response of the peak intensity of the characteristic peak, it can be seen from FIG. 1 that the greater the zearalenone concentration, the weaker the signal.
Fig. 2 is a working curve of SERS quantitative detection of zearalenone molecules by the surface enhanced raman spectroscopy quantitative analysis method of zearalenone according to example 1 of the present invention. The lowest concentration capable of detecting 3 times of signal-to-noise ratio signals is taken as a detection limit, the detection limit is 0.03 mu g/L (S/N is 3), and the linear range and the detection limit of the method can meet the requirement of actual sample analysis.
From fig. 3, the method also uses samples to be detected from different sources to perform SERS detection, and uses aflatoxin B1, fumonisin B1, fumonisin B2, ochratoxin a, ochratoxin B, clavotoxin and zearalenone of the invention as detection objects to perform SERS detection, and as can be seen from fig. 3, the method has good selectivity.
The present invention has been described in detail with reference to the embodiments, but the present invention is not limited to the embodiments described above, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.
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<120> surface enhanced Raman spectroscopy analysis and detection method of zearalenone and application thereof
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Claims (10)
1. A surface enhanced Raman spectroscopy analysis and detection method of zearalenone is characterized by comprising the following steps:
s100, preparing a standard sample: mixing gold nanoparticles, zearalenone aptamer, 3 ', 5, 5' -tetramethylbenzidine and hydrogen peroxide to obtain a premixed solution; mixing the zearalenone standard solutions with different concentrations with the premixed solution to react to obtain zearalenone mixed solutions with different standard concentrations;
s200, taking porous carbon nitride modified by hydrophobic dendritic gold-copper composite nanoparticles as an SERS substrate, obtaining the concentration relation between the peak value of the characteristic Raman displacement position of 3,3 ', 5, 5' -tetramethyl benzidine and zearalenone, and establishing a standard curve;
s300, detecting the concentration of the zearalenone in a sample to be detected: and (3) mixing the sample to be detected with the premixed solution obtained in the step (S100) for reaction to obtain a mixed solution to be detected, detecting the peak value of the characteristic Raman shift of the 3,3 ', 5, 5' -tetramethylbenzidine according to the detection method same as that in the step (S200), and calculating the concentration of the zearalenone in the mixed solution to be detected according to the standard curve in the step (S200).
2. The method of claim 1, wherein the sequence of the zearalenone aptamer is 5'-GAT GGG GAA AGG GTC CCC CTG GGT TGG AGC ATC GGA CA-3'.
3. The method for detecting zearalenone through surface-enhanced raman spectroscopy according to claim 1, wherein the average particle size of the gold nanoparticles is 30 to 70 nm.
4. The method for detecting zearalenone by surface-enhanced raman spectroscopy according to claim 1, wherein the concentration of the zearalenone aptamer is 10 to 1000 nmol/L.
5. The method for detecting zearalenone by surface enhanced raman spectroscopy according to claim 1, wherein the concentration of 3,3 ', 5, 5' -tetramethylbenzidine is 1-20 mg/L.
6. The method for detecting zearalenone by surface-enhanced raman spectroscopy according to claim 1, wherein the concentration of the hydrogen peroxide is 1 to 500 μ M.
7. The method for detecting zearalenone by surface-enhanced raman spectroscopy according to claim 1, wherein in step S200, the porous carbon nitride substrate modified by the hydrophobic dendritic gold-copper composite nanoparticles is prepared by the following method:
s210, adding a gold source, a copper source and a reducing agent into a porous carbon nitride solution, carrying out solvothermal reaction, and adding a hydrophobic reagent for continuous reaction;
s220, filtering the product in the step S210, and distributing the filtered solid in a solid support to obtain a hydrophobic solid SERS substrate; the solid support is chromatographic paper and/or filter paper.
8. The method of claim 7, wherein the concentration of the porous carbon nitride solution in step S210 is 1.42-4.27 mg/mL.
9. The method of claim 7, wherein the temperature of the solvothermal reaction in step S210 is 95-105 ℃.
10. The use of the surface-enhanced raman spectroscopy method of zearalenone according to any one of claims 1 to 9 for the detection of zearalenone in food products.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103954764A (en) * | 2014-05-16 | 2014-07-30 | 华南师范大学 | Method for rapidly and quantitatively determining zearalenone |
| CN109752371A (en) * | 2018-12-28 | 2019-05-14 | 南京邮电大学 | For Hg2+The colorimetric of detection/SERS bimodulus probe preparation method and application |
| US20200292538A1 (en) * | 2016-03-23 | 2020-09-17 | Agency For Science, Technology And Research | Surface enhanced raman spectroscopy (sers) microfluidics biosensor for detecting single and/or multiple analytes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103954764A (en) * | 2014-05-16 | 2014-07-30 | 华南师范大学 | Method for rapidly and quantitatively determining zearalenone |
| US20200292538A1 (en) * | 2016-03-23 | 2020-09-17 | Agency For Science, Technology And Research | Surface enhanced raman spectroscopy (sers) microfluidics biosensor for detecting single and/or multiple analytes |
| CN109752371A (en) * | 2018-12-28 | 2019-05-14 | 南京邮电大学 | For Hg2+The colorimetric of detection/SERS bimodulus probe preparation method and application |
Non-Patent Citations (2)
| Title |
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| SHUMIN SUN，ET AL.: "Colorimetric zearalenone assay based on the use of an aptamer and of gold nanoparticles with peroxidase-like activity" * |
| YA-NING WANG，ET AL.: "A SERS substrate of mesoporous g-C3N4 embedded with in situ grown gold nanoparticles for sensitive detection of 6-thioguanine" * |
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