CN115060704B - Method for detecting methylmercury and aflatoxin B1 by surface enhanced Raman scattering - Google Patents
Method for detecting methylmercury and aflatoxin B1 by surface enhanced Raman scattering Download PDFInfo
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Abstract
The invention discloses a method for detecting methyl mercury and aflatoxin B1 by surface-enhanced Raman scattering, which is based on dual activities of mimic enzyme and Raman enhancement of iron-doped chapter amine based carbon dot modified gold and silver nano, wherein colorless recessive malachite green is oxidized into malachite green with surface-enhanced Raman spectrum activity in the presence of hydrogen peroxide, and the addition of methyl mercury enhances the peroxidase activity of gold nano; establishing a new method for detecting the methyl mercury and the aflatoxin B1 with high sensitivity and selectivity based on the linear relationship between the methyl mercury concentration and the aflatoxin B1 concentration and the SERS increase, wherein the detection limit is 0.1 mug/kg respectively; the method has the characteristics of simple operation, high sensitivity, rapidness and the like.
Description
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a method for simultaneously detecting methylmercury and aflatoxin B1 by surface enhanced Raman scattering.
Background
Mercury and compounds thereof are widely existed in nature, the toxicity of mercury is closely related to the existing form of mercury, the toxicity of organic mercury is higher than that of inorganic mercury, and the toxicity of methyl mercury is the largest in organic mercury. The inorganic mercury can generate methyl mercury under the action of organism alkylation, and the mercury and the methyl mercury are absorbed by passive plants and then are enriched by food chainThe enrichment factor can reach 10 when entering human body 6 ～10 7 . The method for measuring methyl mercury (high performance liquid chromatography-atomic fluorescence combined method, high performance liquid chromatography-inductively coupled plasma mass spectrometry) in the national food safety standard GB 5009.17-2017 determination of total mercury and organic mercury in food. Mycotoxins are secondary toxic metabolites generated under certain environmental conditions in the growth and reproduction process of mold, and the toxins generally have immunotoxicity, teratogenic toxicity, carcinogenic toxicity and the like, and are extremely harmful. Therefore, relevant regulations on mycotoxins in food are promulgated by the U.S. Food and Drug Administration (FDA), the european union (Eu), and relevant departments such as japanese inspection and quarantine.
The instrumental analysis detection method has great advantages in the aspects of detection sensitivity and accuracy, but cannot be used for on-site and rapid detection of the methylmercury and the aflatoxin B1 due to the limitations of factors such as expensive equipment requirements, complex sample pretreatment process, long detection period and the like. Surface-enhanced Raman spectroscopy (SERS) greatly enhances a Raman signal of a target substance by using a precious metal material such as gold and silver, so that detection of a trace substance is realized. However, the existing preparation methods of the Raman reinforcing agent are few, targets capable of generating Raman signals are limited, and SERS application is not wide.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting methyl mercury (MeHg) and aflatoxin B1 (AFB 1) by surface-enhanced Raman scattering, which is based on double activities of mimic enzyme and Raman increase of iron-doped chapter amino carbon point (Fe, OA/CDs) modified gold and silver nano, wherein colorless recessive malachite green is oxidized into malachite green with surface-enhanced Raman spectrum (SERS) activity in the presence of hydrogen peroxide, and the peroxidase activity of the modified gold and silver nano is enhanced by adding methyl mercury, so that the Raman activity is increased due to the formation of gold and silver amalgam, and when aflatoxin B1 exists, the gold and silver amalgam can interact with the methyl mercury to enhance the activity of the nano enzyme, thereby enhancing the oxidation of the recessive malachite green and causing the SERS signal to be increased; establishing a new method for detecting methyl mercury and aflatoxin B1 with high sensitivity and strong selectivity based on the linear relationship between the concentration of methyl mercury and the concentration of aflatoxin B1 and the increase of SERS, wherein the detection limit is 0.1 mug/kg; the method is applied to detection and analysis of methyl mercury and aflatoxin B1 in food, and the result conforms to the relevant national standard determination method; the method has the characteristics of simple operation, high sensitivity, high speed and the like.
The method for detecting the methyl mercury and the aflatoxin B1 by the surface enhanced Raman scattering comprises the following steps:
1. doping the P-type metal oxide with Fe, OA/CDs, H 2 O 2 And sodium citrate as a reducing agent and a stabilizing agent to prepare Fe, OA/CDs modified gold and silver nano (Au, agNPs);
2. adding Fe, OA/CDs modified gold and silver nano Au and AgNPs solution into methyl mercury standard solution, reacting to generate Au and Ag-Hg, and adding recessive malachite green (LMG) solution and H 2 O 2 Generating green malachite green; performing Raman spectrum detection on the mixture by using a portable Raman instrument, and determining 1614cm according to the molecular structure of malachite green and the attribution of Raman peak positions -1 The characteristic peak is used as a discrimination basis for detecting the methyl mercury by the surface enhanced Raman scattering spectrum, and the linear relation between the concentration of the methyl mercury and the peak area of the characteristic peak is determined;
adding Fe, OA/CDs modified gold and silver nano Au and AgNPs solution into methyl mercury solution, reacting to generate Au and Ag-Hg, and adding recessive malachite green (LMG) solution and H 2 O 2 Generating green malachite green, adding aflatoxin B1 standard solution, performing Raman spectrum detection on the mixture with a portable Raman spectrometer, and determining 797cm according to malachite green molecular structure and Raman peak position attribution -1 The characteristic peak is used as a discrimination basis for detecting the aflatoxin B1 by the surface enhanced Raman scattering spectrum, and the linear relation between the concentration of the aflatoxin B1 and the peak area of the characteristic peak is determined;
3. mixing the sample solution to be detected containing methyl mercury with Fe, OA/CDs modified gold and silver nano Au, agNPsMixing, adding recessive malachite green and H 2 O 2 After the reaction, performing Raman spectrum detection by using a portable Raman instrument, and calculating the concentration of the methyl mercury in the sample liquid to be detected according to the peak area of the characteristic peak;
taking a sample solution to be detected containing aflatoxin B1, mixing and reacting the sample solution with Fe, OA/CDs modified gold and silver nano Au, agNPs and methyl mercury solution, and then adding recessive malachite green and H 2 O 2 After the reaction, performing Raman spectrum detection by using a portable Raman instrument, and calculating the concentration of aflatoxin B1 in the sample solution to be detected according to the peak area of the characteristic peak.
The Fe, OA/CDs modified gold and silver nano Au and AgNPs are prepared as follows:
(1) Weighing 1.0-2.0g of octopamine hydrochloride, 0.5-1.0g of citric acid, 0.5-1.0g of ethylenediamine and 0.1-0.5g of ferric chloride, dissolving in 40-60mL of ultrapure water, ultrasonically mixing uniformly, transferring the solution into a polytetrafluoroethylene lining hydrothermal reaction kettle, heating at the constant temperature of 180-200 ℃ for 8-10h, and naturally cooling to room temperature after the reaction is finished to obtain a brown solution; removing large particle impurities from the brown solution with a 0.22 μm filter membrane, centrifuging, and vacuum drying the supernatant to obtain Fe-doped octopamine carbon dots (Fe, OA/CDs);
(2) Mixing 12-15mg of Fe, OA/CDs, 100mmol/L of H 2 O 2 Dissolving 45-55 μ L sodium citrate 20-25mg in 40-60mL ultrapure water, and adding HAuCl 4 ，HAuCl 4 The concentration of the mixed solution is 10-15mg/mL, the mixture is stirred for 5-10 minutes, and AgNO is added 3 ，AgNO 3 The concentration of the iron-doped octopamine carbon-point modified gold and silver nano particles in the mixed solution is 5-10mg/mL, the mixture is stirred for 20-30 minutes in a dark place and centrifuged, and the supernatant is the iron-doped octopamine carbon-point modified gold and silver nano particles (Au, agNPs).
The centrifugation is carried out at 8000-10000 r/min for 10-15min.
The concentration of the methyl mercury standard solution in the detection is 0.22-18.18 mug/L, and the concentration of the aflatoxin B1 standard solution is 0.22-18.18 mug/L; fe, OA/CDs modified gold and silver nano Au, wherein the concentration of the AgNPs solution is 0.1mg/mL, and the addition amount is 50-100 mu L; the concentration of the LMG solution is 100mmol/L, and the addition amount is 10-20 mu L; h 2 O 2 The concentration of (A) is 100mmol/L, and the dosage is 50-100 muL.
The Raman spectrum detection is that the surface enhanced Raman scattering spectrum peak of the methylmercury and aflatoxin B1 standard solution has 797cm when the standard solution is scanned for 10 seconds under the conditions of 785nm exciting light and 500mW laser power -1 ，1184 cm -1 And 1614cm -1 And (4) peak.
The invention has the advantages and technical effects that:
1. the invention adopts iron-doped chapter amino carbon dots and H 2 O 2 And sodium citrate as a reducing agent and a stabilizing agent to prepare the modified gold and silver nano Au, the prepared Fe, OA/CDs modified gold and silver nano Au, agNPs have double activities of mimic enzyme and Raman increase in H 2 O 2 The oxidized colorless recessive malachite green has malachite green with Surface Enhanced Raman Spectroscopy (SERS) activity, the addition of methyl mercury enhances the peroxidase activity of the modified gold-silver nano-material, and the addition of the methyl mercury enhances the Raman activity simultaneously due to the formation of gold-silver amalgam, so that when aflatoxin B1 exists, the activity of the modified gold-silver nano-material can be enhanced by interaction with the methyl mercury, thereby enhancing the oxidation of the recessive malachite green, leading to the rise of SERS signals, having high selectivity for enhancing the SERS signals of the methyl mercury, and establishing a novel SERS detection method of the methyl mercury and the aflatoxin B1;
2. the SERS detection method of the methyl mercury and the aflatoxin B1, which is established by the invention, has high detection sensitivity, the detection limits of the methyl mercury and the aflatoxin B1 reach 0.1 mu g/L, and the coexisting Hg 2+ Other mycotoxins do not interfere with the determination, and the method has good selectivity;
3. the modified gold and silver nano material prepared by the method can form Au-Ag-Hg alloy with methyl mercury, so that the activity of the pseudoperoxidase is increased, aggregation-induced plasmon displacement is caused, and the amplification of Raman signals is realized, so that the detection method has the characteristics of high sensitivity, strong specificity, simplicity in operation, quickness and the like.
Drawings
FIG. 1 shows (Au, agNPs + LMG + H) in example 1 of the present invention 2 O 2 ）、（Au,AgNPs +MeHg+LMG+H 2 O 2 ) And (Au, agNPs + MeHg + LMG + H) 2 O 2 + AFB 1) ultraviolet absorption SpectrumDrawing;
FIG. 2 shows (Au, agNPs + LMG + H) in example 1 of the present invention 2 O 2 ）、（Au,AgNPs +MeHg+LMG+H 2 O 2 ) And (Au, agNPs + MeHg + LMG + H) 2 O 2 SERS plot of + AFB 1);
FIG. 3 is a SERS graph of different concentrations of methylmercury standard solutions in example 1 of the present invention;
FIG. 4 is a linear relationship diagram of the Raman signal intensity and the methylmercury standard solutions with different concentrations in example 1 of the present invention;
FIG. 5 is a SERS graph of aflatoxin B1 standard solutions of various concentrations in example 1 of the present invention;
FIG. 6 is a linear relationship diagram of the aflatoxin B1 standard solutions of different concentrations in example 1 of the present invention with Raman signal intensity;
FIG. 7 shows the results of an interference experiment with different substances in the detection system of the present invention;
FIG. 8 shows the results of the interference experiment of different aflatoxins and other substances on the detection system of the present invention.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1: determination of aflatoxin B1 in peanut sample and methylmercury in edible fungi
1. Preparation of iron-doped octopamine-based carbon dot modified gold nano-particles
(1) Weighing 1.0g of octopamine hydrochloride, 0.5g of citric acid, 0.5g of ethylenediamine and 0.1g of ferric chloride, dissolving in 40mL of ultrapure water, ultrasonically mixing uniformly, transferring the solution into a polytetrafluoroethylene lining hydrothermal reaction kettle, heating at the constant temperature of 180 ℃ for 10 hours, and naturally cooling to room temperature after the reaction is finished to obtain a brown solution; removing large particle impurities from the brown solution with a 0.22 μm filter membrane, centrifuging at 8000r/min for 15min, collecting supernatant, and vacuum drying to obtain iron-doped octopamine carbon dots (Fe, OA/CDs);
(2) 12mg of Fe, OA/CDs, 100mmol/L H 2 O 2 Dissolving 50 μ L and 20mg sodium citrate in 40mL ultrapure water, adding HAuCl 4 ，HAuCl 4 The concentration in the mixed solution is 10mg/mL, the mixture is stirred for 8 minutes, and AgNO is added 3 ，AgNO 3 The concentration of the iron-doped octopamine carbon-point modified gold and silver nano (Au, agNPs) in the mixed solution is 5mg/mL, the mixture is stirred for 20 minutes in a dark place, and the mixture is centrifuged for 15 minutes at 8000r/min, and the supernatant is the iron-doped octopamine carbon-point modified gold and silver nano (Au, agNPs);
2. determination of SERS spectrum detection wave number of methyl mercury and SERS analysis of standard substance
Adding 200 mu L of methyl mercury standard solution and 50 mu L of 0.1mg/mL Au, agNPs solution into a sample bottle, vortex mixing for 30 seconds, reacting for 5 minutes, and then adding 100mmol/L H 2 O 2 10 mu L of 20 mu L of 100mmol/L LMG solution, 0.22-18.18 mu g/L of methyl mercury standard solution, reacting for 10 minutes at room temperature, scanning for 10 seconds under the conditions of 785nm exciting light and 500mW laser power, performing Raman spectrum detection on the mixed solution by using a portable Raman instrument, and performing ultraviolet detection on the enzyme activity of the modified gold-silver nanometer;
the ultraviolet detection result is shown in figure 1, and it can be seen from the figure that the modified gold and silver nano-particles can enhance the ultraviolet absorption signal under the action of methyl mercury, and when aflatoxin B1 exists, aflatoxin B1 can interact with methyl mercury to further enhance the ultraviolet absorption signal of the modified gold and silver nano-particles;
the detection result of Raman spectrum is shown in figure 2, and the SERS spectrogram of methylmercury is observed to be 797cm -1 、1184 cm -1 And 1614cm -1 Determining 1614cm according to the linear correlation between methyl mercury and SERS characteristic peak -1 The characteristic peak is used as the detection basis for detecting methyl mercury by SERS spectrum; the peak intensities of the characteristic peaks in the SERS spectrum of methylmercury varied with the standard solution concentrations (0.22, 1.13, 2.27, 4.54, 6.81, 9.09, 11.36, 14.77, 15.90, 17.04, and 18.18 μ g/L), as shown in fig. 3; the concentration of methyl mercury and the peak area of the characteristic peak are 1614cm -1 The linear regression equation is: y = 12435.97x+55653.59 2 =0.9861 (fig. 4); and all have obvious Raman spectrum peaks when the concentration is as low as 0.1 mug/L; therefore, the detection concentration of the methyl mercury standard solution by the method reaches 0.1 mu g/L.
3. Determination of SERS spectrum detection wave number of aflatoxin B1 and SERS analysis of standard substance
Adding 10 mu L of 5 mu g/L methyl mercury solution, 0.1mg/mL Au, 50 mu L of AgNPs solution and 200 mu L of aflatoxin B1 standard solution into a sample bottle, reacting for 5 minutes, and then adding 100mmol/L H 2 O 2 Reacting 10 mu L of 20 mu L of 100mmol/L LMG solution at room temperature for 10 minutes, scanning for 10s under the conditions of 785nm exciting light and 500mW laser power, and performing Raman spectrum detection on the mixture by using a portable Raman spectrometer; the characteristic peaks are consistent with those in step 2, and the peak intensities of the characteristic peaks in the SERS spectrum of the aflatoxin B1 are changed along with the concentrations of standard solutions (0.22, 1.13, 2.27, 4.54, 6.81, 9.09, 11.36, 14.77, 15.90, 17.04 and 18.18 mu g/L), as shown in FIG. 5; the concentration of aflatoxin B1 and the peak area of the characteristic peak are 797cm -1 The linear regression equation is: y = 9855.67x +54732.53 2 =0.9922 (fig. 6); and all the components still have obvious Raman spectrum peaks when the concentration is as low as 0.1 mu g/L; therefore, the detection concentration of the standard solution of aflatoxin B1 by the method reaches 0.1 mug/L.
4. Determination of methyl mercury content in edible fungi
(1) The sample pretreatment method comprises the following steps: weighing 0.2g of ground and ground edible fungus sample into a glass tube for extraction, putting a magnetic stirrer, adding 10mL of extract (0.07 mol/L HCl), and extracting for 15min at 55 ℃, 15MPa and 110W by using a microwave extractor; standing in refrigerator for 5min after extraction, transferring supernatant into centrifuge tube, centrifuging at 4 deg.C for 5min at 14 000r/min, and sucking supernatant to obtain sample extractive solution;
(2) Measuring methyl mercury in a sample: adding 0.1mg/mL of Au and 50 muL of AgNPs solution into 200 muL of sample extracting solution, reacting for 5 minutes, and then adding 100mmol/L of H 2 O 2 20 μ L of 100mmol/L LMG 10 μ L, reacting at room temperature for 10min, scanning with 785nm excitation light and laser power of 500mW for 10s, and measuring 1614cm by using portable Raman spectrometer -1 And (3) performing Raman spectrum detection, substituting the acquired Raman signal intensity into the regression equation in the step (2), and calculating the concentration of the methyl mercury in the edible fungi to be undetected.
5. Determination of aflatoxin B1 content in peanuts
(1) The sample pretreatment method comprises the following steps: accurately weighing 2g of crushed and homogenized peanut sample, accurately weighing 0.0001g, adding 20mL of methanol aqueous solution with volume concentration of 70%, mixing for 1 minute by vortex, carrying out ultrasonic extraction for 20 minutes in 50 +/-2 ℃ water bath, centrifuging for 10 minutes at 4000r/min, taking out the upper layer solution, repeatedly extracting for 2 times, combining the upper layer solution, putting into a rotary evaporator, and carrying out rotary evaporation until the volume of the solvent is 1-2mL to obtain a sample extracting solution;
(2) Adding 10 mu L of 5 mu g/L methyl mercury standard solution, 0.1mg/mL Au and 50 mu L of AgNPs solution into 200 mu L of sample extracting solution, reacting for 5 minutes, and then adding 100mmol/L H 2 O 2 20 μ L of 100mmol/L LMG solution 10 μ L, reacting at room temperature for 10min, scanning with 785nm excitation light and laser power of 500mW for 10s, and measuring 797cm by using portable Raman spectrometer -1 And (4) carrying out Raman spectrum detection, substituting the acquired Raman signal intensity into the regression equation in the step (3), and calculating to obtain the concentration of the aflatoxin B1 in the peanuts of 3.14 mug/kg.
Example 2: determination of aflatoxin B1 in corn sample and methylmercury in carp sample
1. Preparing iron-doped stamping amino carbon point modified gold nanoparticles:
(1) Weighing 1.5g of octopamine hydrochloride, 0.75g of citric acid, 0.75g of ethylenediamine and 0.3g of ferric chloride, dissolving in 50mL of ultrapure water, ultrasonically mixing uniformly, transferring the solution into a polytetrafluoroethylene lining hydrothermal reaction kettle, heating at the constant temperature of 190 ℃ for 9 hours, and naturally cooling to room temperature after the reaction is finished to obtain a brown solution; removing large particle impurities from brown solution with 0.22 μm filter membrane, centrifuging at 10000r/min for 10min, and vacuum drying the supernatant to obtain iron-doped octopamine carbon dots (Fe, OA/CDs);
(2) 13mg of Fe, OA/CDs, 100mmol/L H 2 O 2 Dissolving 52 μ L and 22mg sodium citrate in 50mL ultrapure water, adding HAuCl 4 ，HAuCl 4 The concentration in the mixed solution is 12mg/mL, the mixture is stirred for 5 minutes, and AgNO is added 3 ，AgNO 3 The concentration of the solution in the mixed solution is 8mg/mL, the solution is stirred for 25 minutes in a dark place and centrifuged for 10 minutes at 10000r/min, and the supernatant is the iron-doped octopamine carbon-point modified gold-silver nanometer（Au,AgNPs）；
2. The linear regression equation of the methylmercury standard solution is determined to be the same as that of example 1;
3. the same linear regression equation of the standard solution of aflatoxin B1 was determined as in example 1;
4. method for measuring content of methyl mercury in carp
(1) Carp sample extraction: weighing 10g (accurate to 0.001 g) of carp and 50mL of 5mol/L hydrochloric acid solution in a 100mL plastic centrifuge tube with a cover, extracting for 60 minutes in an ultrasonic water bath at room temperature, centrifuging for 15 minutes at 4 ℃ at 8000r/min, taking out supernatant, slowly dropwise adding an ammonia water solution (prepared by mixing ammonia water and water in a volume ratio of 1;
(2) Sample extraction: oscillating and extracting a sample extracting solution twice by using 20mL of dichloromethane, oscillating for 10 minutes each time, standing for 10 minutes, collecting and combining dichloromethane extracting solutions into a 50mL colorimetric tube, accurately adding 2mL of back-extraction solution (aqueous solution containing 1% of cysteine and 0.8% of ammonium acetate) by using a graduated pipette for extraction, oscillating for 5 minutes, standing for 10 minutes, and sucking an upper-layer aqueous solution to obtain a sample solution to be detected;
(3) Measuring methyl mercury in a sample: adding 0.1mg/mL Au and 50 muL AgNPs solution into 200 muL of sample liquid to be detected, reacting for 5 minutes, and then adding 100mmol/L H 2 O 2 20 μ L of 100mmol/L LMG 10 μ L, reacting at room temperature for 10min, scanning with 785nm excitation light and laser power of 500mW for 10s, and measuring 1614cm by using portable Raman spectrometer -1 Performing Raman spectrum detection, substituting the collected Raman signal intensity into the regression equation in the step 2, and calculating the concentration of methyl mercury in the carp to be 4.02 mu g/kg;
5. determination of aflatoxin B1 content in corn
(1) The sample pretreatment method comprises the following steps: accurately weighing 2g of a crushed corn sample, accurately weighing 0.0001g, adding 20mL of methanol aqueous solution with volume concentration of 70%, mixing for 1 minute in a vortex manner, carrying out ultrasonic extraction for 20 minutes in a water bath at 50 +/-2 ℃, centrifuging for 10 minutes at 4000r/min, taking out an upper layer solution, repeatedly extracting for 2 times, combining the upper layer solution, putting into a rotary evaporator, and carrying out rotary evaporation until the volume of a solvent is 1-2mL to obtain a sample extracting solution;
(2) Determination of aflatoxin B1 in samples: taking 200 mu L of sample liquid to be detected, adding 10 mu L of 5 mu g/L methyl mercury standard solution, 0.1mg/mL Au and 50 mu L of AgNPs solution, reacting for 5 minutes, and then adding 100mmol/L H 2 O 2 20 μ L of 100mmol/L LMG solution 10 μ L, reaction at room temperature for 10min, scanning at 785nm with excitation light and laser power of 500mW for 10s, and measuring by portable Raman spectrometer at 797cm -1 And (4) performing Raman spectrum detection, substituting the acquired Raman signal intensity into the regression equation in the step (3), and not detecting the aflatoxin B1 in the corn.
Example 3: determination of Mercuric methyl in shrimp and aflatoxin B1 in peanut oil
1. Preparing iron-doped octopamine carbon dot modified gold nanoparticles:
(1) Weighing 2.0g of octopamine hydrochloride, 1.0g of citric acid, 1.0g of ethylenediamine and 0.5g of ferric chloride, dissolving in 60mL of ultrapure water, ultrasonically mixing uniformly, transferring the solution into a polytetrafluoroethylene lining hydrothermal reaction kettle, heating at the constant temperature of 200 ℃ for 8 hours, and naturally cooling to room temperature after the reaction is finished to obtain a brown solution; removing large particle impurities from the brown solution with a 0.22 μm filter membrane, treating at 9000r/min for 12min, and vacuum drying the supernatant to obtain iron-doped octopamine carbon dots (Fe, OA/CDs);
(2) Mixing 15mg of Fe, OA/CDs, 100mmol/L H 2 O 2 Dissolving 50 μ L and 25mg sodium citrate in 60mL ultrapure water, adding HAuCl 4 ，HAuCl 4 The concentration of the solution in the mixed solution is 15mg/mL, the solution is stirred for 10 minutes, and AgNO is added 3 ，AgNO 3 Stirring the mixture for 30 minutes in a dark place with the concentration of 10mg/mL in the mixed solution, and treating the mixture for 12 minutes at 9000r/min to obtain supernatant, namely the iron-doped octopamine carbon-point modified gold-silver nano (Au, agNPs);
2. the same linear regression equation of the methylmercury standard solution was determined as in example 1;
3. the same linear regression equation of the standard solution of aflatoxin B1 was determined as in example 1;
4. determination of methyl mercury content in shrimp sample
(1) Shrimp sample was prepared in the same manner as the carp sample of example 2;
(2) Measuring methyl mercury in a sample: in the same manner as in example 2, the concentration of methylmercury in shrimp was 1.35. Mu.g/kg;
5. determination of aflatoxin B1 content in peanut oil
(1) The sample pretreatment method comprises the following steps: accurately weighing 2g of peanut oil to 0.0001g, adding 1g of NaCl and 5mL of 80 vol% methanol aqueous solution, mixing for 1 minute by vortex, and quantitatively filtering with filter paper to obtain a sample extract;
(2) Adding 10 mu L of 5 mu g/L methyl mercury standard solution and 50 mu L of 0.1mg/mL Au and AgNPs solution into 200 mu L of sample extracting solution, reacting for 5 minutes, and then adding 100mmol/L H 2 O 2 20 μ L of 100mmol/L LMG solution 10 μ L, reacting at room temperature for 10min, scanning with 785nm excitation light and laser power of 500mW for 10s, and measuring 797cm by using portable Raman spectrometer -1 And (4) carrying out Raman spectrum detection, substituting the intensity of the collected Raman signal into the regression equation in the step (3), and calculating the concentration of the aflatoxin B1 in the peanut oil to be 2.02 mu g/kg.
Recovery rate and precision experiment of the detection sample in the above example: respectively adding 2 methylmercury and aflatoxin B1 standard solutions with different concentrations into the detection samples of the embodiments 1-3; each concentration is measured in parallel for 3 times, the standard recovery rate is calculated, and the relative standard deviation RSD is calculated, and the result is shown in tables 1 and 2; the measured standard recovery rate of the methylmercury is 92.1-97.0 percent, the RSD is 3.89-5.76 percent, the standard recovery rate of the aflatoxin B1 is 91.7-94.9 percent, the RSD is 3.74-5.93 percent, and the method has good accuracy and precision;
TABLE 1 sample methylmercury recovery with RSD (n = 3)
TABLE 2 sample aflatoxin B1 recovery on standard and RSD (n = 3)
Method specificity investigation: the methyl mercury is replaced by other substances, the influence of the other substances on the modified gold-silver nano-particles in the reaction system is detected, the concentration of the methyl mercury is 10 mug/L, and FIG. 7 shows other interference substances (FeCl) 3 、CoCl 2 、CdCl 2 、Cr(NO 3 ) 3 、NiCl 2 、AgCl、PbCl 2 、HgCl 2 And Hg (NO) 3 ) 2 Etc.) on the detection system, the concentration of the interference substance is 10 mug/L, and as can be seen from the figure, the methyl mercury SERS system has better selection specificity, and other substances have no enhancing activity effect on the modified gold and silver nano-particles.
In general, AFB1 coexists with AFB2, AFG1 and AFG2 in a ratio of 1.
The aflatoxin B1 and the methyl mercury determination method established by the invention have the advantages of few processing steps, short used time, low processing cost, simple and convenient operation, no need of large-scale instruments and equipment and stronger advantage in actual detection.
Claims (5)
1. A method for detecting methylmercury and aflatoxin B1 by surface enhanced Raman scattering is characterized by comprising the following steps:
(1) Doping Fe, OA/CDs, H with Fe 2 O 2 And sodium citrate is used as a reducing agent and a stabilizing agent to prepare Fe, OA/CDs modified gold and silver nano Au and AgNPs;
(2) Adding Fe, OA/CDs into the methyl mercury standard solutionModifying gold and silver nano Au and AgNPs solution, reacting to generate gold and silver amalgam, and adding recessive malachite green solution and H 2 O 2 Generating green malachite green; performing Raman spectrum detection on the mixture by using a portable Raman instrument, and determining 1614cm according to the molecular structure of malachite green and the attribution of Raman peak positions -1 The characteristic peak is used as a discrimination basis for detecting the methyl mercury by the surface enhanced Raman scattering spectrum, and the linear relation between the concentration of the methyl mercury and the peak area of the characteristic peak is determined;
adding Fe, OA/CDs modified gold and silver nano Au, agNPs solution and aflatoxin B1 standard solution into methyl mercury solution, reacting to generate gold and silver amalgam, and adding recessive malachite green solution and H 2 O 2 Generating green malachite green, performing Raman spectrum detection on the mixture by using a portable Raman instrument, and determining 797cm according to the molecular structure and Raman peak position attribution of the malachite green -1 The characteristic peak is used as a discrimination basis for detecting the aflatoxin B1 by the surface enhanced Raman scattering spectrum, and a linear relation between the concentration of the aflatoxin B1 and the peak area of the characteristic peak is determined;
(3) Taking a sample solution to be detected containing methyl mercury, carrying out mixed reaction with Fe, OA/CDs modified gold and silver nano Au and AgNPs, and then adding recessive malachite green and H 2 O 2 After the reaction, performing Raman spectrum detection by using a portable Raman instrument, and calculating the concentration of the methyl mercury in the sample liquid to be detected according to the peak area of the characteristic peak;
taking a sample solution to be detected containing aflatoxin B1, mixing and reacting the sample solution with Fe, OA/CDs modified gold and silver nano Au, agNPs and methyl mercury solution, and then adding recessive malachite green and H 2 O 2 After the reaction, performing Raman spectrum detection by using a portable Raman instrument, and calculating the concentration of aflatoxin B1 in the sample solution to be detected according to the peak area of the characteristic peak.
2. The method of claim 1, wherein the Fe, OA/CDs modified gold and silver nano Au, agNPs are prepared as follows:
(1) Weighing 1.0-2.0g of octopamine hydrochloride, 0.5-1.0g of citric acid, 0.5-1.0g of ethylenediamine and 0.1-0.5g of ferric chloride, dissolving in 40-60mL of ultrapure water, ultrasonically mixing uniformly, transferring the solution into a polytetrafluoroethylene lining hydrothermal reaction kettle, heating at the constant temperature of 180-200 ℃ for 8-10h, and naturally cooling to room temperature after the reaction is finished to obtain a brown solution; removing large particle impurities from the brown solution by using a 0.22-micron filter membrane, performing high-speed centrifugation, and performing vacuum drying on the supernatant to obtain Fe-doped octopamine carbon dots Fe, OA/CDs;
(2) 12-15mg of iron doped with octopamine carbon dots Fe, OA/CDs and 100mmol/L H 2 O 2 Dissolving 45-55 μ L and 20-25mg sodium citrate in 40-60mL ultrapure water, and adding HAuCl 4 ，HAuCl 4 The concentration of the mixed solution is 10-15mg/mL, agNO is added after the mixed solution is stirred for 5-10 minutes 3 ，AgNO 3 The concentration of the iron-doped octopamine carbon-point modified gold and silver nano Au and AgNPs in the mixed solution is 5-10mg/mL, the mixture is stirred for 20-30 minutes in a dark place and centrifuged, and the supernatant is the iron-doped octopamine carbon-point modified gold and silver nano Au and AgNPs.
3. The method of claim 1, wherein: the concentration of the methylmercury standard solution is 0.22-18.18 mug/L, and the concentration of the aflatoxin B1 standard solution is 0.22-18.18 mug/L; fe, OA/CDs modified gold and silver nano Au, wherein the concentration of the AgNPs solution is 0.1mg/mL, and the addition amount is 50-100 mu L; the concentration of the recessive malachite green solution is 100mmol/L, and the addition amount is 10-20 mu L; h 2 O 2 The concentration of (b) is 100mmol/L, and the dosage is 20-50 μ L.
4. The method of claim 1, wherein: the Raman spectrum detection is performed by scanning for 10s under the conditions of 785nm exciting light and 500mW laser power.
5. The method of claim 2, wherein: the centrifugation is carried out at 8000-10000 r/min for 10-15min.
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