CN114034681B - Surface-enhanced Raman spectrum analysis and detection method for zearalenone and application thereof - Google Patents
Surface-enhanced Raman spectrum analysis and detection method for zearalenone and application thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 238000004458 analytical method Methods 0.000 title claims abstract description 24
- 238000000479 surface-enhanced Raman spectrum Methods 0.000 title claims abstract description 15
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- DAEYIVCTQUFNTM-ABAIWWIYSA-N ochratoxin B Chemical compound C([C@H](NC(=O)C1=CC=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 DAEYIVCTQUFNTM-ABAIWWIYSA-N 0.000 description 1
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Classifications
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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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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a surface-enhanced Raman spectrum analysis and detection method of zearalenone and application thereof. The surface-enhanced Raman spectrum 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 premix; s200, taking porous carbon nitride modified by hydrophobic dendritic gold-copper composite nano particles as an SERS substrate, acquiring the concentration relation between a peak value at the characteristic Raman displacement of 3,3', 5' -tetramethylbenzidine and zearalenone, and establishing a standard curve; s300, detecting the concentration of zearalenone in the sample to be detected, and calculating the concentration of zearalenone in the mixed solution to be detected according to the standard curve of 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 spectrum analysis and detection method of zearalenone and application thereof.
Background
Zearalenone, also known as F-2 toxin, is a common mycotoxin, and is widely used in various crops such as corn, wheat, barley, oat and the like, and foods produced from the above grains as raw materials, such as corn oil, soy sauce, vinegar and the like. The toxicity of zearalenone is mainly manifested in influencing the reproductive system of human and animals and in higher carcinogenicity and teratogenicity. Therefore, how to judge the zearalenone content in food has important meaning for guaranteeing the food safety. In addition, some illegal merchants use zearalenone-contaminated food materials for production and processing in order to increase profits, which greatly increases the occurrence of zearalenone poisoning events. Therefore, the establishment of a rapid, accurate, stable and sensitive analysis method of zearalenone in foods has great significance.
The detection method of the zearalenone which is widely used at present comprises a thin layer chromatography method, a high performance liquid chromatography method, an enzyme-linked immunosorbent assay method and the like, but the quantitative analysis of the thin layer chromatography method is poor, the specificity of the enzyme-linked immunosorbent assay method is poor, the high performance liquid chromatography method needs to be combined with fluorescence detection, the operation is complex, and the analysis time is long.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems in the prior art. Therefore, the invention provides a surface-enhanced Raman spectrum analysis and detection method of zearalenone.
The second aspect of the invention provides application of the surface-enhanced Raman spectrum analysis detection method of zearalenone in the concentration of zearalenone in corn.
The first aspect of the invention provides a surface-enhanced Raman spectrum analysis and detection method of zearalenone, which comprises the following steps:
s100, preparing a standard sample: mixing gold nanoparticles, zearalenone aptamer, 3', 5' -tetramethylbenzidine and hydrogen peroxide to obtain a premix; mixing and reacting the standard solutions of the zearalenone with different concentrations with the premix solution to obtain mixed solutions of the zearalenone with different standard concentrations;
s200, taking porous carbon nitride modified by hydrophobic dendritic gold-copper composite nano particles as an SERS substrate, acquiring the concentration relation between a peak value at the characteristic Raman displacement of 3,3', 5' -tetramethylbenzidine and zearalenone, and establishing a standard curve;
s300, detecting the concentration of zearalenone in a sample to be detected: and (2) mixing the sample to be detected with the premix of the step (S100) to obtain a mixed solution to be detected, detecting the peak value of the characteristic Raman displacement of the 3,3', 5' -tetramethylbenzidine according to the detection method which is the same as that of the step (S200), and calculating the concentration of zearalenone in the mixed solution to be detected according to the standard curve of the step (S200).
The principle of the invention is as follows: the nano hardware fitting has better catalytic performance, so that 3,3', 5' -tetramethyl benzidine can be catalyzed into 3,3', 5' -tetramethyl benzidine in an oxidation state in the presence of hydrogen peroxide, and the SERS signal is weakened. And the aptamer is wrapped on the surface of the nano gold through electrostatic adsorption, so that the catalysis of the aptamer can be inhibited. When zearalenone exists, the aptamer is subjected to morphological change and is separated from the nanogold due to the strong recognition effect between the aptamer and the zearalenone, and at the moment, the nanogold can catalyze 3,3', 5' -tetramethylbenzidine into 3,3', 5' -tetramethylbenzidine in an oxidation state. Therefore, the higher the zearalenone concentration, the more the aptamer is detached, the better the catalytic performance of the nano gold, the more 3,3', 5' -tetramethylbenzidine in an oxidation state, and the weaker the SERS signal; conversely, the stronger the SERS signal.
The invention relates to a technical scheme of a surface-enhanced Raman spectrum analysis method of zearalenone, which has at least the following beneficial effects:
according to the invention, the porous carbon nitride modified by the hydrophobic dendritic gold-copper composite nano particles is used as a SERS substrate, and the concentration of the zearalenone is determined by the recognition effect between the zearalenone and the zearalenone aptamer, and the method has high selectivity, sensitivity, rapidity and accuracy.
According to some embodiments of the invention, the sample to be tested in step S300 is further subjected to pretreatment.
According to some embodiments of the invention, the sequence of the zearalenone aptamer is 5'-GATGGGGAAAGGGTCCCCCTGGGTTGGAGCATCGGACA-3'.
According to some embodiments of the invention, the gold nanoparticles have an average particle size of 30 to 70nm.
According to some embodiments of the invention, the zearalenone aptamer concentration is between 10 and 1000nmol/L.
According to some embodiments of the invention, the concentration of 3,3', 5' -tetramethylbenzidine is 1 to 20mg/L.
According to some embodiments of the invention, the concentration of hydrogen peroxide is 1 to 500 μm.
According to some embodiments of the present invention, in step S200, the porous carbon nitride substrate modified by the hydrophobic dendritic gold-copper composite nanoparticle is prepared by the following method:
s210, adding a gold source, a copper source and a reducing agent into a porous carbon nitride solution, performing solvothermal reaction, and then adding a hydrophobic reagent to continue the 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 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 a second aspect of the invention, there is provided the use of the surface-enhanced raman spectroscopy method of zearalenone for detecting zearalenone.
Drawings
FIG. 1 is a graph showing SERS response to quantitative analysis of zearalenone molecules using example 1 of the present invention;
FIG. 2 is a working curve of SERS quantitative detection of zearalenone using example 1 of the present invention;
FIG. 3 is a bar graph of SERS responses of various zearalenone analogues.
Detailed Description
The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described with reference to the embodiments, but the present invention is not limited to these embodiments.
Example 1
Embodiment 1 provides a surface-enhanced raman spectrum analysis and detection method of zearalenone, which comprises the following steps of:
s210, adding 7.5mL of 10.0mmol/L chloroauric acid, 7.5mL of 10.0mmol/L cupric chloride and 0.7mL of 1.0 mmol/L glucose solution into 2.84mg/mL porous carbon nitride solution, performing solvothermal reaction, heating to 100 ℃, and preserving heat for 30min; 75.0mg of hexadecylamine was added to continue the reaction;
s220, filtering the product in the step S210, and distributing the filtered solid in a solid support to obtain the SERS substrate of the porous carbon nitride modified by the hydrophobic dendritic gold-copper composite nano particles.
A surface-enhanced Raman spectrum 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 adding 50 mu L of 10 mu M hydrogen peroxide and 100 mu L of 6.0 mg/L3, 3', 5' -tetramethyl benzidine respectively, and fully mixing to obtain a premix; mixing 50 mu L of zearalenone with mass concentration 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 premix, and reacting for 15min to obtain the zearalenone mixed solution with different standard concentrations;
s200, establishing a standard curve: dripping the mixed zearalenone with different standard concentrations into an SERS substrate of porous carbon nitride modified by the prepared hydrophobic dendritic gold-copper composite nano particles, directly detecting the mixed zearalenone with different concentrations by using a DeltaNu 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 with 3,3', 5' -tetramethylbenzidine -1 The peak intensity of the characteristic peak at the site was used for quantification. Establishing a standard curve by using the concentration relation between the peak value at the characteristic Raman displacement and zearalenone;
pretreating a sample to be tested: 40.0g of crushed corn and oat samples are taken in a homogenizing cup, 4g of sodium chloride and 100mL of 90% acetonitrile solution (v/v) are added, and the mixture is stirred and extracted at high speed by a homogenizer for 2min, and quantitative filter paper is used for filtration. Transferring 10.0mL of filtrate, adding 40mL of water for dilution and mixing, and filtering through glass fiber filter paper until the filtrate is clear, wherein the filtrate is ready for use. The immunoaffinity column was connected under a glass syringe, 10.0mL of the filtrate was accurately removed, and injected into the glass syringe. An air pressure pump was connected to the glass syringe and the pressure was adjusted to allow the solution to pass through the immunoaffinity column slowly at a flow rate of 1 to 2 drops/s until some air entered the affinity column. The column was rinsed 1 time with 5mL of water at a flow rate of 1 to 2 drops/s until some air had entered the affinity column and all effluent was discarded. 1.5mL of methanol was accurately added for elution at a flow rate of about 1 drop/s. Collecting eluate in glass test tube, blow-drying with nitrogen below 55deg.C, dissolving residue with 1.0mL ultrapure water, filtering with 0.22 μm microporous membrane, and collecting filtrate.
S300, detecting the concentration of zearalenone in a sample to be detected: respectively taking 50 mu L of the processed sample to be detected, mixing with the premix solution in the step S100, reacting for 15min to obtain a mixed solution to be detected, detecting the peak value at the characteristic Raman displacement of 3,3', 5' -tetramethyl benzidine according to the detection method same as the step S200, and calculating 1330cm of 3 data -1 And substituting the peak average value and the relative standard deviation into a zearalenone concentration standard curve to obtain the corn, wherein the zearalenone concentration in the oat sample is 4.6 (+ -0.17) mug/kg and 8.8 (+ -0.55) mug/kg respectively.
In order to verify the accuracy of the SERS detection method of the zearalenone in the embodiment 1 on the zearalenone detection, the solution to be detected in the embodiment 4 is subjected to labeling treatment, a labeled sample is obtained by adding 1.0 mug/kg, 20.0 mug/kg and 200.0 mug/kg of zearalenone standard solution to a sample to be detected, then the SERS detection is carried out for 3 times, peak average value and relative deviation at 1330cm < -1 > of 3 data are calculated, the peak average value and the relative deviation are substituted into a zearalenone concentration standard curve, so that the zearalenone concentration in the labeled sample is obtained, and the labeled recovery rate of the sample is calculated to be 97.5-102.9%, and the relative standard deviation is calculated to be 3.4-8.2%.
The accuracy of detection by the established SERS analysis method was verified by high performance liquid chromatography mass spectrometry (HPLC-MS). The sample liquid to be measured after the treatment was subjected to HPLC-MS measurement, and a high performance liquid chromatograph-mass spectrometer was supplied by Shimadzu corporation, and the selected column was a C18 column (50 mm. Times.2.0 mm,2 μm). The mobile phase adopts acetonitrile-water, gradient elution is adopted, and the elution conditions are as follows: 0-5min: mobile phase a increased from 25% to 70%;5-6min: mobile phase a was maintained at 70%;6-9min: mobile phase a decreased from 70% to 25%. The flow rate was 0.2mL/min, and the sample injection amount was 5. Mu.L. The mass spectrum conditions are as follows: ionization mode, electrospray ionization (ESI-); capillary voltage 3.0kV; the source temperature is 120 ℃; the desolvation temperature is 350 ℃; taper hole airflow: nitrogen gas with the flow rate of 100L/h; solvent-free gas flow: nitrogen gas at a flow rate of 600L/h; collision gas: argon, collision air pressure is 2.60 multiplied by 10 < -4 > Pa; scanning mode: scanning negative ions; parent ion (m/z): 317.1; daughter ion (m/z): 174.9/273.9. The HPLC-MS detection shows that the concentration of zearalenone in the oat sample is 4.4 (+ -0.17) mug/kg and 9.1 (+ -0.38) mug/kg respectively, and the relative deviation between the corn and the SERS analysis method is less than 5.2%, thus proving the reliability of the analysis method.
Example 2
This example 2 is identical to the starting material and assay of example 1, except that the assay sample source is two different corn oils and 1330cm of 3 data are calculated -1 The peak value average value and the relative standard deviation are substituted into a zearalenone concentration standard curve, so that the zearalenone concentration in 2 corn oil samples is 137.0 (+ -4.80) mug/kg and 115.6 (+ -6.01) mug/kg respectively.
To verify the accuracy of the SERS assay of zearalenone of example 5 on zearalenone detection, the test solution of example 4 was subjected to a labeling treatment in which 1.0. Mu.g/kg, 20.0. Mu.g/kg and 200.0. Mu.g/kg of a standard solution of zearalenone were added to the sample, followed by SERS assay for 3 consecutive times and 1330cm of 3 data were calculated -1 And substituting the peak average value and the relative deviation into a standard curve of the concentration of the zearalenone to obtain the concentration of the zearalenone in the standard sample, and calculating to obtain the standard recovery rate of the sample of 102.3-108.8% and the relative standard deviation of 3.2-7.3%.
The accuracy of detection by the established SERS analysis method was verified by high performance liquid chromatography mass spectrometry (HPLC-MS). The sample liquid to be measured after the treatment was subjected to HPLC-MS measurement, and a high performance liquid chromatograph-mass spectrometer was supplied by Shimadzu corporation, and the selected column was a C18 column (50 mm. Times.2.0 mm,2 μm). The mobile phase adopts acetonitrile-water, gradient elution is adopted, and the elution conditions are as follows: 0-5min: mobile phase a increased from 25% to 70%;5-6min: mobile phase a was maintained at 70%;6-9min: mobile phase a decreased from 70% to 25%. The flow rate was 0.2mL/min, and the sample injection amount was 5. Mu.L. The mass spectrum conditions are as follows: ionization mode, electrospray ionization (ESI-); capillary voltage 3.0kV; source temperature: 120 ℃; the desolvation temperature is 350 ℃; taper hole airflow: nitrogen gas with the flow rate of 100L/h; solvent-free gas flow: nitrogen gas at a flow rate of 600L/h; collision gas: argon, collision air pressure is 2.60 multiplied by 10 < -4 > Pa; scanning mode: scanning negative ions; parent ion (m/z): 317.1; daughter ion (m/z): 174.9/273.9. The HPLC-MS detection shows that the concentration of zearalenone in the oat sample is 135.1 (+ -0.54) mug/kg and 113.5 (+ -0.79) mug/kg respectively, and the relative deviation between the corn and the SERS analysis method is less than 1.8%, which proves that the analysis method has good accuracy.
FIG. 1 shows 1330cm of zearalenone and 3,3', 5' -tetramethylbenzidine in different concentrations according to example 1 of the invention -1 From the response plot of peak intensities at characteristic peaks, 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 using the surface-enhanced Raman spectrum quantitative analysis method of zearalenone in example 1 of the present invention. The lowest concentration of the signal with the signal to noise ratio of 3 times can be detected is taken as the detection limit, the detection limit is 0.03 mug/L (S/N=3), and the linear range and the detection limit of the method can meet the requirement of actual sample analysis.
According to the invention, as shown in fig. 3, SERS detection is carried out by using samples to be detected from different sources, and by using aflatoxin B1, fumonisin B2, ochratoxin A, ochratoxin B, and zearalenone according to the invention as detection objects, SERS detection is carried out, and as shown in 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, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Sequence listing
<110> university of Zhongshan
<120> surface enhanced Raman spectrum analysis and detection method of zearalenone and application thereof
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Claims (5)
1. The surface-enhanced Raman spectrum analysis and detection method of zearalenone is characterized by comprising the following steps of:
s100, preparing a standard sample: mixing gold nanoparticles, zearalenone aptamer, 3', 5' -tetramethylbenzidine and hydrogen peroxide to obtain a premix; mixing and reacting the standard solutions of the zearalenone with different concentrations with the premix solution to obtain mixed solutions of the zearalenone with different standard concentrations;
s200, taking porous carbon nitride modified by hydrophobic dendritic gold-copper composite nano particles as an SERS substrate, acquiring the concentration relation between a peak value at the characteristic Raman displacement of 3,3', 5' -tetramethylbenzidine and zearalenone, and establishing a standard curve;
s300, detecting the concentration of zearalenone in a sample to be detected: mixing a sample to be detected with the premix of the step S100 to obtain a mixed liquid to be detected, detecting a peak value at a characteristic Raman displacement position of 3,3', 5' -tetramethylbenzidine according to the detection method which is the same as that of the step S200, and calculating the concentration of zearalenone in the mixed liquid to be detected according to a standard curve of the step S200;
the sequence of the zearalenone aptamer is 5'-GAT GGG GAA AGG GTC CCC CTG GGT TGG AGC ATC GGA CA-3';
the average particle size of the gold nanoparticles is 30-70 nm;
in step S200, the porous carbon nitride substrate modified by the hydrophobic dendritic gold-copper composite nanoparticle is prepared by the following method:
s210, adding a gold source, a copper source and a reducing agent into a porous carbon nitride solution, performing solvothermal reaction, and then adding a hydrophobic reagent to continue the reaction; the gold source is from chloroauric acid; the copper source is from copper chloride and/or copper nitrate;
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;
the concentration of the porous carbon nitride solution in the step S210 is 1.42-4.27 mg/mL;
the solvothermal reaction temperature in the step S210 is 95-105 ℃.
2. The method for detecting zearalenone by surface-enhanced Raman spectrum analysis according to claim 1, wherein the concentration of zearalenone aptamer is 10-1000 nmol/L.
3. The method for detecting zearalenone by surface-enhanced Raman spectroscopy according to claim 1, wherein the concentration of 3,3', 5' -tetramethylbenzidine is 1-20 mg/L.
4. The method for detecting zearalenone by surface-enhanced Raman spectroscopy according to claim 1, wherein the concentration of hydrogen peroxide is 1-500 mu M.
5. The use of the surface-enhanced raman spectroscopy detection method of zearalenone according to any one of claims 1-4 for detecting zearalenone in food.
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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 |
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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 |
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.《Microchimica Acta》.2018,第185卷第1-7页. * |
| 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.《Sensors and Actuators B: Chemical》.2018,第260卷第400-407页. * |
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