CN113218927B - Method for detecting acrylamide content in fried food based on aptamer fluorescent probe - Google Patents

Method for detecting acrylamide content in fried food based on aptamer fluorescent probe Download PDF

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CN113218927B
CN113218927B CN202110541964.7A CN202110541964A CN113218927B CN 113218927 B CN113218927 B CN 113218927B CN 202110541964 A CN202110541964 A CN 202110541964A CN 113218927 B CN113218927 B CN 113218927B
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邹小波
甘子玉
石吉勇
胡雪桃
李文亭
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Abstract

The invention belongs to the technical field of food safety detection, and particularly relates to a method for detecting the content of acrylamide in fried food by using an aptamer fluorescent probe. The method comprises the following steps: dissolving zirconium chloride and 2-amino terephthalic acid in DMF, performing ultrasonic treatment, heating, cooling, washing and drying to obtain a material, dissolving the material in Tris-HCl buffer solution, adding FAM-ssDNA, performing vortex mixing, and reacting at room temperature to obtain an aptamer fluorescence sensor; the aptamer fluorescent sensor prepared by the invention can specifically identify acrylamide, so that the detection of the acrylamide content in fried food is realized; the invention has simple operation, rapid and accurate detection, is suitable for the detection of real food samples, and is beneficial to ensuring the quality and safety of food.

Description

Method for detecting acrylamide content in fried food based on aptamer fluorescent probe
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a method for detecting the content of acrylamide in fried food by using an aptamer fluorescent probe.
Technical Field
Acrylamide is a harmful organic compound that can cause genetic toxicity, muscle weakness, neurotoxicity, sensory loss, and carcinogenicity. In 1994, acrylamide has been classified as a second carcinogen by the world health organization international agency for research on cancer. People can come into contact with acrylamide through the skin, mucous membranes, respiratory tract and digestive tract, wherein diet is an important way for people to come into contact with acrylamide. Many foods cooked by high temperature cooking contain high levels of acrylamide, particularly when processed into foods rich in starch and protein, such as potatoes, rice, sorghum, and the like, which can affect the health of people after long-term ingestion. Therefore, it is necessary to detect the acrylamide content in food rapidly and accurately to ensure food safety and health of product consumers.
At present, the detection of acrylamide mainly depends on traditional technologies including high performance liquid chromatography, gas chromatography-mass spectrometry, enzyme-linked immunosorbent assay and the like. Although the methods can accurately detect the content of acrylamide, the methods often have certain limitations due to the complex pretreatment method, long time consumption, high cost, need of professional skills and the like, and are not favorable for realizing rapid detection. In recent years, fluorescence sensing technology has attracted much attention because of its simplicity and short time consumption, but currently, there are few studies on the detection of acrylamide by fluorescence sensing technology. Therefore, there is an urgent need to design and develop a fluorescent probe to achieve sensitive detection of acrylamide in fried foods.
Disclosure of Invention
In view of the deficiencies of the prior art, the present invention is directed to solving one of the problems; the invention develops an aptamer fluorescent probe, can realize low-cost, high-sensitivity and specific detection of acrylamide in fried food, and overcomes the defects of complex operation, long time consumption, complex preparation and the like of the existing acrylamide detection technology.
In order to achieve the above object, the present invention specifically comprises the following steps:
step 1, dissolving zirconium chloride and 2-amino terephthalic acid in N, N-Dimethylformamide (DMF), performing ultrasonic treatment and heating, cooling reactants, washing and drying to obtain a yellow metal organic framework material, and marking the yellow metal organic framework material as UiO-66-NH 2 A material;
step 2, the UiO-66-NH prepared in the step 1 2 Dissolving the material in a Tris-HCl buffer solution, adding a 6-carboxyl fluorescein dye modified acrylamide fluorescence aptamer chain solution (FAM-ssDNA solution), vortex uniformly mixing, and reacting at room temperature to obtain an aptamer fluorescence sensor solution, wherein the aptamer fluorescence sensor solution is marked as an MOF-aptamer fluorescent probe solution;
step 3, establishing a linear regression equation for detecting acrylamide: preparing acrylamide solutions with different concentrations, adding the acrylamide solutions with different concentrations into the MOF-aptamer fluorescent probe solution prepared in the step 2, correspondingly adding one MOF-aptamer fluorescent probe solution into one acrylamide solution with one concentration, standing for reaction, and respectively adding a complementary strand solution (csDNA solution) of an acrylamide fluorescent aptamer chain modified by 6-carboxyl fluorescein dye into the solution to obtain a mixed solution for incubation at room temperature; finally, placing the mixed solution in a fluorescence spectrophotometer to measure the fluorescence intensity value I of the mixed solution 520 Establishing a linear regression method for detecting acrylamide according to the relation between the detected fluorescence intensity value and the corresponding acrylamide concentration log (c)A process;
and 4, detecting and analyzing acrylamide in the food sample: pretreating a sample according to the national standard GB 5009.204-2014 determination of acrylamide in food-stable isotope dilution liquid chromatography-mass spectrometry/mass spectrometry to obtain a sample extracting solution; then adding the sample extracting solution into the prepared MOF-aptamer fluorescent probe solution, standing for reaction at room temperature, then adding csDNA solution, incubating at room temperature, and measuring the fluorescence intensity value I of the mixed solution in a fluorescence spectrophotometer 520 And substituting the fluorescence intensity value of the sample into the acrylamide linear regression equation established in the step S1 to calculate the concentration of the acrylamide in the sample.
Preferably, the ratio of the zirconium chloride, the 2-aminoterephthalic acid and the N, N-dimethylformamide used in the step 1 is 0.133 to 0.164g: 0.098-0.115 g: 25-40 mL.
Preferably, the power of the ultrasound in the step 1 is 60-100W, and the time is 5-10 min; the heating temperature is 90-120 ℃, and the time is 10-24 h; the washing is respectively washing for 1 to 3 times by using N, N-dimethylformamide and ethanol solution; the drying temperature is 60-80 ℃, and the drying time is 24-48 h.
Preferably, the base sequence of the 6-carboxyfluorescein dye-modified acrylamide aptamer chain (FAM-ssDNA) in the step 2 is 5'-FAM-ACC GCA TCA TGC CGA AAG GAC TAC CGG AAA CGG CAA ATC CTC G-3'; the concentration of the FAM-ssDNA solution is 5-10 mu M.
Preferably, the UiO-66-NH described in step 2 2 The dosage ratio of the material, the acrylamide fluorescent aptamer chain solution modified by 6-carboxyl fluorescein dye and the Tris-HCl buffer solution is 0.1-0.5 g: 100-200 μ L: 2-10 mL; the concentration of the Tris-HCl buffer solution is 10mM, and the pH value is 7.4.
Preferably, the vortex mixing time in the step 2 is 1-2 min; the reaction time is 20-60 min.
Preferably, the concentration range of the acrylamide solution with different concentrations in the step 3 is 50 nM-10 μ M; the volume ratio of the acrylamide solution to the MOF-aptamer fluorescent probe solution when mixed is 1.
Preferably, the standing time in the step 3 is 30-60 min, and the incubation time is 60-90 min.
Preferably, the base sequence of the complementary strand of the acrylamide fluorescent aptamer chain modified by the 6-carboxyl fluorescein dye in the step 3 is 5; the concentration of the csDNA solution is 5-10 mu M, and the volume ratio of the acrylamide solution to the csDNA solution in the mixed solution is 1.
Preferably, the slit width of the fluorescence spectrophotometer in step 3 is 0.5nm, the excitation wavelength of the detection mixed solution is 480nm, the wavelength range is 508-600 nm, and the fluorescence intensity value at 520nm is detected.
Preferably, the volume ratio of the sample extracting solution, the MOF-aptamer fluorescent probe solution and the solution of the complementary strand of the acrylamide fluorescent aptamer chain modified by the 6-carboxyl fluorescein dye in the step 4 is 1:1:1.
advantageous technical effects
(1) The aptamer fluorescent probe prepared by the invention can realize the specificity detection of acrylamide, so that the result is more accurate and reliable.
(2) The synthetic method of the aptamer fluorescent probe prepared by the invention is simple and convenient to operate, and the detection of acrylamide can be realized without professional technicians.
(3) The aptamer fluorescent probe prepared by the invention can specifically detect acrylamide with the concentration range of 50 nM-10 μ M, the detection limit is 15.6nM, and the aptamer fluorescent probe has a wider linear detection range and a lower detection limit, and has higher detection precision and sensitivity than the traditional high-performance liquid chromatography.
(4) The invention passes UiO-66-NH for the first time 2 Preparing an aptamer fluorescent probe for specifically detecting acrylamide by combining a metal organic framework material with an aptamer chain of acrylamide; compared with the prior art, the method has the advantages of convenient operation, high stability and high sensitivity; fluorescent aptamers are adsorbed to UiO-66-NH under the action of van der Waals' force 2 Surface, by UiO-66-NH 2 Photoinduced electron transfer with fluorescent aptamersQuenching the fluorescence of the fluorescent aptamer by shift effect; when acrylamide is present, acrylamide binds to the fluorescent aptamer, and when the complementary strand of the fluorescent aptamer is added to the solution, the aptamer that fails to bind to acrylamide binds to the complementary strand and is separated from UiO-66-NH 2 The surface is detached, and the fluorescence of the fluorescence aptamer after detachment is recovered. In addition, the acrylamide fluorescent aptamer chain designed by the invention can specifically recognize acrylamide, and the acrylamide can be detected only by using the fluorescent aptamer probe. The invention has the advantages of fine design and simple operation, is suitable for detecting real food samples, and is beneficial to ensuring the quality and safety of food.
Drawings
FIG. 1 shows UiO-66-NH in example 1 2 TEM images of fluorescent materials.
FIG. 2 is a graph of fluorescence spectra of the aptamer fluorescent probe of example 1 with different concentrations of acrylamide and specific concentrations of csDNA.
FIG. 3 is a graph showing the fluorescence intensity values I of aptamer fluorescent probes 520 A linear regression curve (excitation wavelength 480 nm) was plotted against the log (c) of the acrylamide concentration.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail with reference to the drawings and the following detailed description, but the scope of the present invention is not limited thereto.
The 6-carboxyl fluorescein dye modified acrylamide fluorescence aptamer chain (FAM-ssDNA) and the complementary chain (csDNA) used in the invention are all from the company of biological engineering (Shanghai).
Example 1:
to further illustrate the present invention, an aptamer fluorescent probe for acrylamide was prepared and used for detecting acrylamide in potato chips as an example, and the specific steps are as follows:
step 1, preparing UiO-66-NH 2 Materials: dissolving 0.133g of zirconium chloride and 0.115g of 2-amino terephthalic acid in 36.6mL of DMF, ultrasonically dispersing for 10min, then placing the mixture into a 100mL polytetrafluoroethylene reaction kettle, reacting for 12h at 120 ℃, cooling to room temperature, and respectively using DMF and ethanol solutionWashing for 3 times, and drying at 60 ℃ for 24 hours to obtain UiO-66-NH 2 A material; prepared UiO-66-NH as shown in FIG. 1 2 The probe is an octahedron with clear outline, and the side length of the particle is within the range of 250-400 nm.
Step 2, preparing an aptamer fluorescent probe solution: 0.2g of UiO-66-NH was taken 2 Dissolving in 10mL Tris-HCl buffer solution (10 mM, pH 7.4), adding 200. Mu.L FAM-ssDNA solution with concentration of 5. Mu.M, mixing for 1min by vortex, and standing at room temperature for reaction for 60min to obtain MOF-aptamer fluorescent probe solution;
step 3, establishing a standard method for detecting acrylamide: preparing acrylamide solutions with concentrations of 0nM, 50nM, 100nM, 200nM, 500nM, 1. Mu.M, 2. Mu.M, 5. Mu.M and 10. Mu.M with Tris-HCl buffer solution (10 mM, pH 7.4), respectively, and adding 200. Mu.L of the acrylamide solution to 200. Mu.L of the prepared MOF-aptamer fluorescent probe solution, allowing to stand for 30min, followed by adding 200. Mu.L of csDNA solution with a concentration of 5. Mu.M, and incubating at room temperature for 60min; after the incubation is finished, putting the solution into a fluorescence spectrophotometer to obtain fluorescence spectrograms under different concentrations according to the concentration of acrylamide solutions of 50nM, 100nM, 200nM, 500nM, 1 muM, 2 muM, 5 muM and 10 muM and corresponding fluorescence intensity values I 520 And establishing a linear regression equation for detecting acrylamide.
FAM-ssDNA showed a fluorescence emission peak (520 nm) for 6-carboxyfluorescein under 480nm excitation. In UiO-66-NH 2 In the presence of the material, FAM-ssDNA is adsorbed on UiO-66-NH due to van der Waals' force 2 Surface of material due to UiO-66-NH 2 A photoinduced electron transfer phenomenon between the material and 6-carboxyfluorescein (FAM) so that the fluorescence of the FAM is quenched; after acrylamide is added, the acrylamide can be specifically reacted with guanine N 7 The FAM-ssDNA is still adsorbed on the UiO-66-NH after the binding 2 A material surface; in this case, when CSDNA, which is a complementary strand of FAM-ssDNA, is added to the solution, CSDNA is bound to FAM-ssDNA and is expressed as UiO-66-NH 2 The surface is separated, and the fluorescence of the separated FAM-ssDNA and the FAM-ssDNA in the csDNA compound is recovered; FAM-ssDNA bound to acrylamide cannot bind to csDNA and is derived from UiO-66-NH 2 The surface is detached and thus the fluorescence of FAM-ssDNA cannot be recovered. The specific detection of acrylamide can be realized through the change of the fluorescence intensity of FAM.
According to the fluorescence intensity value I of the solution 520 A curve is fitted to the change in acrylamide concentration, the curve corresponding to a function of: y = -640.7X +3132.8, correlation coefficient R 2 =0.991, linear range 0.05-10 μ M.
Step 4, detecting and analyzing acrylamide in the potato chip sample: pretreating a sample according to the national standard GB 5009.204-2014 determination of acrylamide in food-stable isotope dilution liquid chromatography-mass spectrometry/mass spectrometry; crushing the potato chip sample, weighing 2g of the potato chip sample, filtering, performing ultrasonic treatment and the like to obtain an extract containing acrylamide, and metering the volume to 10mL to obtain the acrylamide extract of the potato chip; dripping 200 mu L of potato chip acrylamide extract liquid into 200 mu L of MOF-aptamer fluorescent probe solution, reacting for 30min, adding 200 mu L of csDNA solution with the concentration of 5 mu M, and incubating for 60min at room temperature; after the incubation is finished, the solution is placed in a fluorescence spectrophotometer to obtain the fluorescence intensity value I of the solution 520 And calculating the acrylamide content in the potato chips through the constructed linear regression equation of the acrylamide.
The utility of the method for detecting acrylamide in potato chips was evaluated by a standard recovery method and compared with High Performance Liquid Chromatography (HPLC). The results are shown in table 1, which shows that the method has better consistency with the results measured by high performance liquid chromatography.
TABLE 1 results and recovery of acrylamide in potato chip samples
Figure BDA0003071932270000051
Description of the invention: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and within the scope of the following claims.

Claims (10)

1. A method for detecting the acrylamide content in fried food based on aptamer fluorescence probe is characterized by comprising the following steps:
step 1, dissolving zirconium chloride and 2-amino terephthalic acid in N, N-dimethylformamide, performing ultrasonic treatment and heating, cooling reactants, washing and drying to obtain a yellow metal organic framework material, and marking as UiO-66-NH 2 A material;
step 2, the UiO-66-NH prepared in the step 1 2 Dissolving the material in a Tris-HCl buffer solution, adding an acrylamide fluorescence aptamer chain solution modified by 6-carboxyl fluorescein dye, performing vortex mixing uniformly, and reacting at room temperature to obtain an aptamer fluorescence sensor solution, wherein the aptamer fluorescence sensor solution is marked as an MOF-aptamer fluorescence probe solution; the UiO-66-NH 2 The dosage ratio of the material, the acrylamide fluorescent aptamer chain solution modified by 6-carboxyl fluorescein dye and the Tris-HCl buffer solution is 0.1-0.5 g: 100-200 μ L: 2-10 mL;
step 3, establishing a linear regression equation for detecting acrylamide: preparing acrylamide solutions with different concentrations, adding the acrylamide solutions with different concentrations into the MOF-aptamer fluorescent probe solution prepared in the step 2, correspondingly adding one MOF-aptamer fluorescent probe solution into one acrylamide solution with one concentration, standing for reaction, and respectively adding complementary chain solutions of the acrylamide fluorescent aptamer chains modified by 6-carboxyl fluorescein dye into the solutions to obtain mixed solutions for incubation at room temperature; finally, placing the mixed solution in a fluorescence spectrophotometer to measure the fluorescence intensity value I of the mixed solution 520 Establishing a linear regression equation for detecting acrylamide according to the relation between the detected fluorescence intensity value and the corresponding acrylamide concentration logarithm value;
and 4, detecting and analyzing acrylamide in the food sample: firstly, preparing a sample extracting solution; then adding the sample extract to the prepared MOFStanding the aptamer fluorescence probe solution at room temperature for reaction, adding the csDNA solution, incubating at room temperature, and measuring the fluorescence intensity value I of the mixed solution in a fluorescence spectrophotometer 520 And substituting the fluorescence intensity value of the sample into the acrylamide linear regression equation established in the step S1, and calculating the concentration of the acrylamide in the sample.
2. The method for detecting the acrylamide content in fried foods based on aptamer fluorescence probe as claimed in claim 1, wherein the ratio of the zirconium chloride, 2-aminoterephthalic acid and N, N-dimethylformamide in step 1 is 0.133-0.164 g: 0.098-0.115 g:25 to 40mL.
3. The method for detecting the acrylamide content in the fried food based on the aptamer fluorescence probe as claimed in claim 1, wherein the power of the ultrasound in step 1 is 60-100W, and the time is 5-10 min; the heating temperature is 90-120 ℃, and the time is 10-24 h; the washing is respectively washing for 1 to 3 times by using N, N-dimethylformamide and ethanol solution; the drying temperature is 60-80 ℃, and the drying time is 24-48 h.
4. The method for detecting the acrylamide content in fried foods based on an aptamer fluorescent probe according to claim 1, wherein the base sequence of the 6-carboxyfluorescein dye-modified acrylamide aptamer chain in the step 2 is 5'-FAM-ACC GCA TCA TGC CGA AAG GAC TAC CGG AAA CGG CAA ATC CTC G-3'; the concentration of the acrylamide aptamer chain solution modified by the 6-carboxyl fluorescein dye is 5-10 mu M.
5. The method for detecting the acrylamide content in fried food using aptamer-based fluorescence probe as claimed in claim 1, wherein the concentration of Tris-HCl buffer solution in step 2 is 10mM and the pH is 7.4.
6. The method for detecting the acrylamide content in the fried food based on the aptamer fluorescent probe as claimed in claim 1, wherein the vortex mixing time in the step 2 is 1-2 min; the reaction time is 20-60 min.
7. The method for detecting the acrylamide content in fried foods based on the aptamer fluorescent probe as claimed in claim 1, wherein the concentration of the acrylamide solution with different concentration in step 3 is in the range of 50nM to 10 μ M; the volume ratio of the acrylamide solution to the MOF-aptamer fluorescent probe solution is 1; the standing time is 30-60 min, and the incubation time is 60-90 min.
8. The method for detecting the acrylamide content in fried food based on aptamer fluorescent probe as claimed in claim 1, wherein the base sequence of the complementary strand of the acrylamide fluorescent aptamer chain modified by 6-carboxyfluorescein dye in step 3 is 5 'CGA GGA TTT GCC GTT TCC GGT AGT CCT TTC GGC ATG ATG CGG T-3'; the concentration of the csDNA solution is 5-10 mu M, and the volume ratio of the acrylamide solution to the complementary strand solution of the acrylamide fluorescent aptamer chain modified by 6-carboxyl fluorescent dye in the mixed solution is 1.
9. The method for detecting the acrylamide content in fried food based on aptamer fluorescence probe as claimed in claim 1, wherein the slit width of the fluorescence spectrophotometer in step 3 is 0.5nm, the excitation wavelength of the detection mixed solution is 480nm, the wavelength range is 508-600 nm, and the fluorescence intensity value at 520nm is detected.
10. The method for detecting the acrylamide content in fried foods based on the aptamer fluorescence probe of claim 1, wherein the volume ratio of the sample extract, the MOF-aptamer fluorescence probe solution and the solution of the complementary strand of the acrylamide fluorescence aptamer chain modified by the 6-carboxyfluorescein dye in the step 4 is 1:1:1.
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