CN113218927B - A detection method for acrylamide content in fried food based on aptamer fluorescent probe - Google Patents

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

Detection of acrylamide content in fried food based on aptamer fluorescent probe method

technical field

The invention belongs to the technical field of food safety detection, and in particular relates to a method for detecting acrylamide content in fried food with an aptamer fluorescent probe.

technical background

Acrylamide is a harmful organic compound that can cause genotoxicity, muscle weakness, neurotoxicity, loss of sensation, and carcinogenicity. In 1994, the International Agency for Research on Cancer of the World Health Organization listed acrylamide as a second-class carcinogen. People can be exposed to acrylamide through the skin, mucous membranes, respiratory tract and digestive tract, among which diet is an important way for people to be exposed to acrylamide. Many foods cooked at high temperatures contain high levels of acrylamide, especially processed foods rich in starch and protein, such as potatoes, rice, and sorghum. Long-term intake of such foods can affect people's health. Therefore, it is necessary to quickly and accurately detect the content of acrylamide in food to ensure food safety and the health of product consumers.

At present, the detection of acrylamide mainly relies on traditional techniques including high performance liquid chromatography, gas chromatography-mass spectrometry, and enzyme-linked immunosorbent assay. Although these methods can accurately detect the content of acrylamide, they often have certain limitations due to cumbersome pretreatment methods, long time-consuming, high cost, and professional skills, which are not conducive to rapid detection. In recent years, fluorescent sensing technology has attracted much attention due to its simple operation and short time-consuming, but there are few studies on the detection of acrylamide by fluorescent sensing technology. Therefore, there is an urgent need to design and develop a fluorescent probe for sensitive detection of acrylamide in fried foods.

Contents of the invention

In view of the deficiencies in the prior art, the present invention aims to solve one of the problems; the present invention develops an aptamer fluorescent probe, which can realize low-cost, high-sensitivity and specific detection of acrylamide in fried foods, and overcomes the The existing acrylamide detection technology has disadvantages such as complex operation, long time consumption, and complicated preparation.

In order to achieve the above object, the present invention specifically comprises the following steps:

Step 1. Dissolve zirconium chloride and 2-aminoterephthalic acid in N,N-dimethylformamide (DMF), sonicate and heat. After the reactant is cooled, wash and dry to obtain a yellow metal Organic framework material, denoted as UiO-66-NH ₂ material;

Step 2. Dissolve the UiO-66- _NH2 material prepared in step 1 in Tris-HCl buffer solution, add 6-carboxyfluorescein dye-modified acrylamide fluorescent aptamer chain solution (FAM-ssDNA solution), vortex After mixing, react at room temperature to obtain the aptamer fluorescent sensor solution, which is recorded as MOF-aptamer aptamer fluorescent probe solution;

Step 3, establish a linear regression equation for detecting acrylamide: prepare different concentrations of acrylamide solutions, add different concentrations of acrylamide solutions to the MOF-aptamer aptamer fluorescent probe solution prepared in step 2, a concentration of acrylamide A MOF-aptamer aptamer fluorescent probe solution is added to the amide solution, and the complementary chain solution (csDNA solution) of the acrylamide fluorescent aptamer chain modified by 6-carboxyfluorescein dye is added to the above solution after standing reaction , to obtain the mixed solution and incubate at room temperature; finally place it in a spectrofluorometer to measure the fluorescence intensity value I ₅₂₀ of the above mixed solution. The relationship between, establish the linear regression equation that detects acrylamide;

Step 4, detection and analysis of acrylamide in food samples: according to the national standard "GB 5009.204-2014 Determination of Acrylamide in Food - Liquid Chromatography with Stable Isotope Dilution - Mass Spectrometry/Mass Spectrometry", the sample is pretreated to obtain a sample extract Then the sample extract is added to the prepared MOF-aptamer aptamer fluorescent probe solution, left to stand at room temperature for reaction, and then csDNA solution is added, and after incubation at room temperature, the concentration of the above mixed solution is measured in a fluorescence spectrophotometer. For the fluorescence intensity value I ₅₂₀ , the fluorescence intensity value of the sample is brought into the acrylamide linear regression equation established in step S1 to calculate the concentration of acrylamide in the sample.

Preferably, the dosage ratio of zirconium chloride, 2-aminoterephthalic acid and N,N-dimethylformamide in step 1 is 0.133-0.164g:0.098-0.115g:25-40mL.

Preferably, the ultrasonic power described in step 1 is 60-100W, and the time is 5-10min; the temperature of the heating is 90-120°C, and the time is 10-24h; Methylformamide and ethanol solution are washed 1-3 times respectively; the drying temperature is 60-80° C. and the drying time is 24-48 hours.

Preferably, the base sequence of the 6-carboxyfluorescein dye-modified acrylamide aptamer chain (FAM-ssDNA) described in 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 μM.

Preferably, the amount ratio of the UiO-66- _NH2 material, 6-carboxyfluorescein dye-modified acrylamide fluorescent aptamer chain solution and Tris-HCl buffer solution described in step 2 is 0.1～0.5g:100～ 200 μL: 2-10 mL; the concentration of the Tris-HCl buffer solution is 10 mM, and the pH is 7.4.

Preferably, the vortex mixing time in step 2 is 1-2 min; the reaction time is 20-60 min.

Preferably, the concentration range of the different concentrations of acrylamide solutions in step 3 is 50nM-10μM; the volume ratio of the acrylamide solution mixed with the MOF-aptamer aptamer fluorescent probe solution is 1:1.

Preferably, the resting time in step 3 is 30-60 minutes, and the incubation time is 60-90 minutes.

Preferably, the base sequence of the complementary strand of the 6-carboxyfluorescein dye-modified acrylamide fluorescent aptamer chain described 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 μM, and the volume ratio of the acrylamide solution to the csDNA solution in the mixed solution is 1:1.

Preferably, the slit width of the fluorescence spectrophotometer in step 3 is 0.5 nm, the excitation wavelength for detecting the mixed solution is 480 nm, the wavelength range is 508-600 nm, and the fluorescence intensity value at 520 nm is detected.

Preferably, the volume ratio of the solution of the sample extraction solution, the MOF-aptamer aptamer fluorescent probe solution and the complementary chain of the acrylamide fluorescent aptamer chain modified by a 6-carboxyfluorescein dye in step 4 is 1:1 :1.

Beneficial technical effect

(1) The aptamer fluorescent probe prepared by the present invention can realize the specific detection of acrylamide, making the result more accurate and reliable.

(2) The synthesis method of the aptamer fluorescent probe prepared by the present invention is simple and easy to operate, and the detection of acrylamide can be realized without professional technicians.

(3) The aptamer fluorescent probe prepared by the present invention can specifically detect acrylamide in a concentration range of 50nM～10μM, and the detection limit is 15.6nM, which has a wider linear detection range and a lower detection limit, compared with The detection accuracy and sensitivity of traditional university liquid chromatography are high.

(4) For the first time, the present invention has prepared an aptamer fluorescent probe for specific detection of acrylamide through the combination of UiO-66- _NH2 metal organic framework material and acrylamide aptamer chain; compared with existing inventions, this The invention is easy to operate, has high stability and sensitivity; under the action of van der Waals force, the fluorescent aptamer is adsorbed on the surface of UiO-66-NH ₂ , and the photoinduced electrons between UiO-66-NH ₂ and the fluorescent aptamer The transfer effect quenches the fluorescence of the fluorescent aptamer; when acrylamide exists, acrylamide combines with the fluorescent aptamer, and at this time, after adding the complementary chain of the fluorescent aptamer to the above solution, it fails to combine with the acrylamide The bound aptamer can combine with its complementary chain and detach from the surface of UiO-66-NH ₂ , and the fluorescence of the aptamer after detachment is restored. In addition, the acrylamide fluorescent aptamer chain designed in the present invention can specifically recognize acrylamide, and this fluorescent aptamer probe must be used to realize the detection of acrylamide. The invention has excellent design and simple operation, is applicable to the detection of real food samples, and is beneficial to guarantee the quality and safety of the food.

Description of drawings

FIG. 1 is a TEM image of UiO-66-NH ₂ fluorescent material in Example 1.

FIG. 2 is a fluorescence spectrum diagram of the aptamer fluorescent probe in Example 1 added with different concentrations of acrylamide and specific concentrations of csDNA.

Fig. 3 is a linear regression curve (excitation wavelength 480nm) drawn between the fluorescence intensity value I ₅₂₀ of the aptamer fluorescent probe and the logarithm value log(c) of the acrylamide concentration.

specific embodiment

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

Both the 6-carboxyfluorescein dye-modified acrylamide fluorescent aptamer strand (FAM-ssDNA) and its complementary strand (csDNA) used in the present invention come from Sangon Bioengineering (Shanghai) Co., Ltd.

Example 1:

In order to further illustrate the present invention, it is an example to make an aptamer fluorescent probe for acrylamide and use it for the detection of acrylamide in potato chips. The specific steps are as follows:

Step 1. Preparation of UiO-66-NH ₂ material: Dissolve 0.133g of zirconium chloride and 0.115g of 2-aminoterephthalic acid in 36.6mL of DMF, ultrasonically disperse for 10min and put them into a 100mL polytetrafluoroethylene reactor Inside, react at 120°C for 12h, cool to room temperature, wash with DMF and ethanol solution three times respectively, and dry at 60°C for 24h to obtain UiO-66-NH ₂ material; as shown in Figure 1, the prepared UiO-66-NH ₂ The probe is a well-defined octahedron with side lengths in the range of 250-400 nm.

Step _2. Preparation of aptamer fluorescent probe solution: Dissolve 0.2g UiO-66-NH in 10mL of Tris-HCl buffer solution (10mM, pH 7.4), then add 200μL of FAM-ssDNA solution with a concentration of 5μM , after vortex mixing for 1 min, leave to react at room temperature for 60 min to obtain MOF-aptamer aptamer fluorescent probe solution;

Step 3, establish a standard method for detecting acrylamide: use Tris-HCl buffer solution (10mM, pH 7.4) to prepare acrylamide solutions with concentrations of 0nM, 50nM, 100nM, 200nM, 500nM, 1μM, 2μM, 5μM and 10μM, and Add 200 μL acrylamide solution to the prepared 200 μL MOF-aptamer aptamer fluorescent probe solution, let it stand for 30 minutes, then add 200 μL csDNA solution with a concentration of 5 μM, and incubate at room temperature for 60 minutes; after the incubation, place the above solution in In the fluorescence spectrophotometer, the fluorescence spectra at different concentrations are obtained, and the linearity of the detection of acrylamide is established according to the concentration of 50nM, 100nM, 200nM, 500nM, 1μM, 2μM, 5μM and 10μM acrylamide solution and the corresponding fluorescence intensity value I ₅₂₀ regression equation.

The fluorescence emission peak (520nm) of 6-carboxyfluorescein displayed by FAM-ssDNA under 480nm excitation. In the presence of UiO-66-NH ₂ material, FAM-ssDNA was adsorbed on the surface of UiO-66 _{-NH 2 material} due to the effect of van der Waals force. The phenomenon of photo-induced electron transfer between them makes the fluorescence of FAM quenched; after adding acrylamide, acrylamide can specifically bind to the N ₇ site of guanine, and FAM-ssDNA is still adsorbed on UiO-66 -NH ₂ material surface; at this time, after adding the complementary strand csDNA of FAM-ssDNA to the above solution, the csDNA binds to FAM-ssDNA and detaches from the surface of UiO-66-NH ₂ , and the detached FAM-ssDNA and csDNA complex The fluorescence of FAM-ssDNA in UiO-66-NH 2 was restored; FAM-ssDNA combined with acrylamide could not be combined with csDNA and detached from the surface of UiO-66-NH ₂ , so the fluorescence of FAM-ssDNA could not be restored. The specific detection of acrylamide can be realized by the change of the fluorescence intensity of the FAM.

According to the fitting curve between the fluorescence intensity value I ₅₂₀ of the above solution and the concentration change of acrylamide, the function corresponding to the curve is: Y=-640.7X+3132.8, the correlation coefficient R ² =0.991, and the linear range is 0.05-10 μM.

Step 4. Detection and analysis of acrylamide in French fries samples: Pretreat the samples according to the national standard "GB 5009.204-2014 Determination of Acrylamide in Food-Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry/Mass Spectrometry"; The sample is pulverized, and 2g of French fries sample is weighed, and the extract containing acrylamide is obtained by filtering, ultrasonication and other steps, and the volume is adjusted to 10mL to obtain the French fries acrylamide extract; 200μL of the French fries acrylamide extract is added dropwise In the MOF-aptamer aptamer fluorescent probe solution, add 200 μL csDNA solution with a concentration of 5 μM after reacting for 30 minutes, and incubate at room temperature for 60 minutes; after the incubation, put the above solution in a fluorescence spectrophotometer to obtain its fluorescence intensity value I ₅₂₀ , through the constructed linear regression equation of acrylamide, the content of acrylamide in French fries was calculated.

The utility of this method for the detection of acrylamide in French fries was evaluated by spike recovery and compared with high performance liquid chromatography (HPLC). The results are shown in Table 1, and the results show that this method has good consistency with the results measured by high performance liquid chromatography.

Table 1 Detection results and recovery rate of acrylamide in French fries samples

Explanation: the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the specification has described the present invention in detail with reference to the above-mentioned embodiments, those of ordinary skill in the art should understand , the present invention can still be modified or equivalently replaced; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims (10)

1. A kind of detection method based on aptamer fluorescent probe to acrylamide content in fried food, is characterized in that, comprises the following steps: Step 1, dissolving zirconium chloride and 2-aminoterephthalic acid in N,N-dimethylformamide, ultrasonication and heating, after cooling the reactants, washing and drying to obtain a yellow metal organic framework material , denoted as UiO-66-NH ₂ material; Step 2. Dissolve the UiO-66- _NH2 material prepared in step 1 in Tris-HCl buffer solution, add 6-carboxyfluorescein dye-modified acrylamide fluorescent aptamer chain solution, vortex and mix well and store at room temperature Carry out the reaction to obtain the aptamer fluorescent sensor solution, which is referred to as the MOF _- aptamer aptamer fluorescent probe solution; The dosage ratio of solution and Tris-HCl buffer solution is 0.1~0.5g: 100~200μL: 2~10mL; Step 3, establish a linear regression equation for detecting acrylamide: prepare different concentrations of acrylamide solutions, add different concentrations of acrylamide solutions to the MOF-aptamer aptamer fluorescent probe solution prepared in step 2, a concentration of acrylamide Add a MOF-aptamer aptamer fluorescent probe solution to the amide solution, and add the complementary chain solution of the acrylamide fluorescent aptamer chain modified by 6-carboxyfluorescein dye to the above solution after standing reaction to obtain a mixed solution Incubate at room temperature; finally place in a fluorescence spectrophotometer to measure the fluorescence intensity value I ₅₂₀ of the above mixed solution, and establish the detection of acrylamide according to the relationship between the fluorescence intensity value and the corresponding logarithmic value of the acrylamide concentration. The linear regression equation; Step 4. Detection and analysis of acrylamide in food samples: first prepare the sample extract; then add the sample extract to the prepared MOF-aptamer aptamer fluorescent probe solution, let stand at room temperature for reaction, and then add csDNA solution After incubating at room temperature, measure the fluorescence intensity value I ₅₂₀ of the above mixed solution in a spectrofluorometer, and bring the fluorescence intensity value of the sample into the linear regression equation of acrylamide established in step S1 to calculate the concentration of acrylamide in the sample concentration. 2. the detection method based on aptamer fluorescent probe to acrylamide content in fried food according to claim 1, is characterized in that, zirconium chloride, 2-aminoterephthalic acid and N , The dosage ratio of N-dimethylformamide is 0.133～0.164g: 0.098～0.115g: 25～40mL. 3. The method for detecting acrylamide content in fried food based on aptamer fluorescent probe according to claim 1, characterized in that the ultrasonic power described in step 1 is 60-100W, and the time is 5-100W. 10min; the heating temperature is 90-120°C, and the time is 10-24h; the washing is 1-3 times with N,N-dimethylformamide and ethanol solution respectively; the drying temperature is 60 ～80℃, the time is 24～48h. 4. the detection method based on aptamer fluorescent probe of claim 1 to acrylamide content in fried food, is characterized in that, the acrylamide aptamer of 6-carboxyfluorescein dye modification described in step 2 The base sequence of the chain 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-carboxyfluorescein dye is 5-10μM. 5. the detection method based on aptamer fluorescent probe to acrylamide content in fried food according to claim 1, is characterized in that, the concentration of the Tris-HCl buffer solution described in step 2 is 10mM, and pH is 7.4. 6. the method for detecting acrylamide content in fried food based on aptamer fluorescent probe according to claim 1, is characterized in that, the vortex mixing time described in step 2 is 1～2min; The reaction time is 20-60min. 7. The detection method for acrylamide content in fried food based on aptamer fluorescent probe according to claim 1, characterized in that the concentration range of the different concentrations of acrylamide solutions described in step 3 is 50nM～10μM ; The volume ratio of the acrylamide solution mixed with the MOF-aptamer aptamer fluorescent probe solution is 1:1; the standing time is 30-60 min, and the incubation time is 60-90 min. 8. the method for detecting acrylamide content in fried food based on aptamer fluorescent probe according to claim 1, is characterized in that, the acrylamide fluorescence adaptation of 6-carboxyfluorescein dye modification described in step 3 The base sequence of the complementary strand of the body chain 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 μM, and the acrylamide in the mixed solution The volume ratio of the solution to the complementary chain solution of the 6-carboxyfluorescein dye-modified acrylamide fluorescent aptamer chain is 1:1. 9. the method for detecting acrylamide content in fried food based on aptamer fluorescent probe according to claim 1, is characterized in that, the slit width of fluorescence spectrophotometer described in step 3 is 0.5nm, detects The excitation wavelength of the mixed solution is 480nm, the wavelength range is 508-600nm, and the fluorescence intensity value at 520nm is detected. 10. the detection method based on aptamer fluorescent probe to acrylamide content in fried food according to claim 1, is characterized in that, described in step 4, sample extract, MOF-aptamer aptamer fluorescent probe The volume ratio of the solution and the solution of the complementary chain of the 6-carboxyfluorescein dye-modified acrylamide fluorescent aptamer chain is 1:1:1.

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