CN107505298B - Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe - Google Patents

Abstract

The invention discloses a method for detecting cyromazine in milk based on a G-quadruplet aptamer fluorescent probe. When the detection system does not contain cyromazine, fluorescein FAM modified on the aptamer releases fluorescence at 520 nm; when cyromazine is added into a system, cyromazine is combined with thymine on functional nucleic acid to promote the functional nucleic acid to form a G-quadruplex structure, so that the fluorescence release at 520nm is inhibited, and the quantitative detection of cyromazine in milk can be realized by measuring the fluorescence signal based on that the released fluorescence signal is in inverse proportion to the concentration of cyromazine. The method can be used for detecting the cyromazine in the milk, and has the advantages of high detection sensitivity, low detection limit of 0.68ppb, good selectivity, simple and convenient operation, simple pretreatment and no need of large-scale instruments.

Description

A G-quadruplex nucleic acid aptamer-based fluorescent probe for the detection of cyromazine in milk Methods

technical field

The invention belongs to the technical field of cyromazine detection, in particular to a method for detecting cyromazine in milk based on a G-quadruplex nucleic acid aptamer fluorescent probe.

Background technique

The chemical name of Cyromazine (Cyr) is 2-cyclopropylamino-4,6-diaminotriazine, which belongs to triazine or atrazine, simazine, atrazine and permethazine. Triazepine compound is a kind of high-efficiency insect growth inhibitor and parasitic insecticide, which is used as a feed additive in the process of livestock and poultry breeding. Studies have shown that most of cyromazine enters the body of animals in the form of original drug or metabolites, which are excreted through milk or feces and urine, and then exposed to soil and water environment through livestock manure. Cypromazine in soil or water environment re-enters the food chain through environmental fate, causing potential risks to the health of organisms at different trophic levels and human health. Cyromazine is metabolized to melamine (Mel) by dealkylation in animals and plants, and the main metabolites of melamine are cyanuric acid (CA), cyanuric acid amide (Ammelide). , Amd) and ammeline (Ammeline, Amn). Long-term exposure to melamine can lead to bladder stones and significantly increase the incidence of bladder cancer.

The rapid detection of cyromazine and its metabolites in milk can effectively prevent toxic and harmful substances from entering the human body. Cyromazine and its metabolite melamine are a group of small molecular weight and highly polar compounds. It is very difficult to extract cyromazine and its related metabolites from actual samples. The US EPA strictly stipulates that cyromazine must be used as a feed additive and can only be used in feeding troughs in stables, but there are still flies that can be sprayed for feeding places, composting, garbage, etc. After oral administration of cyromazine, most of it is excreted in the form of original drug or metabolite melamine through animal urine and feces, and there is very little residue in animals. The US EPA and PRC have established the maximum residue standard for cyromazine in a series of animal and plant foods, of which milk is 0.05mg/kg.

According to the limit standard requirements of cyromazine and its metabolite melamine in food, currently commonly used methods for the detection of cyromazine and its metabolite residues include volumetric analysis; chromatographic analysis methods, including high performance liquid chromatography; immunoassay Chemical analysis; optical analysis, etc. In GB 29704-2013, ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine the residues of cyromazine in animal food. With the detection of veterinary drug residues of cyromazine included in the mandatory inspection items for raw milk purchase and dairy products, due to the limitations of the above-mentioned detection methods and conditions, the huge number of tests will lead to huge costs and heavy workload, which is difficult to meet. The demand for rapid on-site detection requires the improvement of existing detection methods and the development of high-throughput, rapid, and highly sensitive detection methods for cyromazine for daily monitoring.

Nucleic acid aptamers are selected from a large number of oligonucleotide libraries with high specificity and high tuberculosis of nucleic acid fragments through exponential enrichment ligand system in vitro evolution (SELEX) technology. However, compared with protein antibodies and biological enzymes, nucleic acid aptamers have higher affinity, stability and specificity, and are easy to label and design sensors. They have been used for nucleic acid, protein, inorganic metal ions, virus particles and cells. detection. Although it has been reported that thymine can bind to melamine, the metabolite of cyromazine, through hydrogen bonding, aptamers that can specifically bind to cyromazine to form G-quadruplexes have not been screened or synthesized. Based on this, the use of fluorescence spectrophotometer for detection has not been reported.

SUMMARY OF THE INVENTION

The object of the present invention is to utilize the cyromazine of different concentrations to quench the phenomenon of the fluorescence intensity of fluorescein in the detection solution to varying degrees, and to provide a kind of detection of cyromazine in the milk with high sensitivity, good selectivity and low cost method.

In order to achieve the above object, the present invention provides a method for detecting cyromazine in milk by using a nucleic acid aptamer detection solution modified by fluorescein FAM that can form a G-quadruplex. Fluorescein FAM is modified on the 5' end of the nucleic acid aptamer to form a stable detection solution. When the excitation light wavelength is 480nm, it will release stable fluorescence at 520nm.

The principle of the invention is: according to the structural characteristics of cyromazine, combined with the property that cyromazine can be combined with thymine through hydrogen bonds, the design and synthesis of thymine is mainly, and guanine-rich cyromazine is high Specific aptamer and modified fluorescein FAM at the 5' end. The presence of cyromazine can change the spatial structure of the aptamer and form a stable G-quadruplex structure. Due to the change of the spatial structure of the aptamer, the distance between the fluorophore attached above and cyromazine becomes smaller, thereby quenching the fluorescence. Through the relationship between the concentration of cyromazine and the fluorescence quenching rate, that is, the value of the quenching rate F ₀ /F has a linear relationship with the concentration of cyromazine within a certain range, so by analyzing the fluorescence quenching rate F ₀ /F The change of cyromazine can realize the quantitative detection of cyromazine in milk.

The technical solutions of the present invention are specifically introduced as follows.

The invention provides a method for detecting cyromazine in milk based on a G-quadruplex nucleic acid aptamer fluorescent probe. The specific steps are as follows:

(1) at the temperature of 24～26 ℃, after mixing 2min, obtain that the cyromazine content is maintained at A standard solution between 0-10ppb; wherein: the detection solution is obtained by diluting a FAM-modified nucleic acid aptamer with Tris-acetic acid buffer, and the FAM-modified nucleic acid aptamer is 5'-FAM-GGTTGGTTGGTTGGTTTT-3 ;

(2) get the standard solution prepared above, place in the cuvette, after setting the excitation wavelength to be 480nm, measure its fluorescence intensity at 520nm with a fluorescence spectrophotometer; there is the fluorescence that cyromazine measures in the detection system The intensity is denoted as F, the fluorescence intensity measured in the absence of cyromazine is denoted as F ₀ , and the quenching rate (F ₀ -F)/F ₀ is calculated;

(3) cyromazine of different concentrations and corresponding quenching rate (F ₀ -F)/F ₀ are plotted, and standard curve is drawn;

(4) After pre-treatment of the milk sample to be detected, mix it with the detection solution of the same concentration in step (1), fully mix and react for 1-3 minutes, and use a fluorescence spectrophotometer to test the mixture at an excitation wavelength of 480 nm at 520 nm. The fluorescence intensity of , and then calculate its quenching rate (F ₀ -F)/F ₀ ;

(5) According to the quenching rate (F ₀ -F)/F ₀ of the milk sample to be detected, check the standard curve to obtain the content of cyromazine in the milk sample to be detected.

In the present invention, the concentration of the detection solution is 20-30 nmol/L.

In the present invention, the steps of the pretreatment of the milk sample are as follows: the steps of the pretreatment of the milk sample are as follows: first, mix 4-6 mL of 1wt% acetic acid and 1.0 mL of the milk sample, and let stand for 5 minutes; then 8000-12000 r/min Centrifuge for 8-12 minutes, take the supernatant, and adjust the pH to 8.0 with 1.5-2.5 mol/L NaOH.

Compared with the prior art, the present invention has the beneficial effects that the detection means provided by the method has high sensitivity and good selectivity, the lowest detection limit is 0.68ppb, the operation is simple, the pretreatment is simple, no large-scale instrument is required, and the detection method can be used for cyclopropylamine in milk. detection of oxazine.

Description of drawings

Figure 1 is a schematic diagram of the principle of rapid detection of cyromazine based on G-quadruplex nucleic acid aptamer fluorescent probe.

Figure 2 is a circular dichroism chromatogram of the detection system.

Figure 3 is a graphical representation of the effect of temperature on the detection system.

Figure 4 is a graphical representation of the effect of reaction time and K ⁺ on the detection system.

Fig. 5 is the experimental result of system selectivity.

Figure 6 is the relationship between cyromazine concentration and fluorescence quenching ratio (F ₀ -F)/F ₀ .

Detailed ways

The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

Figure 1 is a schematic diagram of the principle of rapid detection of cyromazine based on G-quadruplex nucleic acid aptamer fluorescent probe. When there is no cyromazine in the detection system, the fluorescein FAM modified on the aptamer will release fluorescence at 520 nm; when cyromazine is added to the system, thymine on cyromazine and functional nucleic acid Combined, it promotes the formation of G-quadruplex structure of functional nucleic acid, and inhibits the fluorescence release at 520nm. Based on the fact that the released fluorescence signal is inversely proportional to the concentration of cyromazine, the quantitative detection of cyromazine in milk can be realized by measuring the fluorescence signal. .

Example 1

(1) System circular dichroism detection: prepare two centrifuge tubes, add nucleic acid aptamers respectively, mix the nucleic acid aptamers with cyromazine, and then scan by circular dichroism at the wavelength of 220nm ~ 320nm to prove that G-tetra Formation of conjoined structures. Figure 2 is a circular dichroism chromatogram of the detection system.

(2) The influence of temperature on the detection system: prepare a mixed system of cyromazine detection solution with the final concentrations of cyromazine as 0ppb, 6ppb, 15ppb, 40ppb, 100ppb and 200ppb respectively, set the excitation wavelength to 480nm and use fluorescence spectrophotometry The fluorescence intensity at 520nm was measured by meter at 25℃, 35℃, 45℃ and 55℃, respectively. The fluorescence intensity measured in the presence of cyromazine in the detection system is recorded as F, and the fluorescence intensity measured in the absence of cyromazine is recorded as F ₀ , and F ₀ /F is calculated. Figure 3 is a graph showing the effect of temperature on the detection system, and the temperature shown in the figure is optimal at 25°C.

(3) The influence of K ⁺ on the detection system: respectively prepare 0, 10, 50, 100ppb K ⁺ solutions, mix with 25nM nucleic acid aptamer, and set the excitation wavelength during the reaction time with the detection solution for 0 to 360 seconds. The fluorescence intensity at 520 nm was measured with a spectrofluorometer for 480 nm. The fluorescence intensity measured in the presence of K ⁺ in the detection system is recorded as F, and the fluorescence intensity measured in the absence of K ⁺ is recorded as F ₀ , and the quenching rate (F ₀ -F)/F ₀ is calculated. Figure 4 shows the effect of K ⁺ on the detection system. The increase of K ⁺ concentration does not affect the fluorescence quenching rate of the system.

(4) Influence of reaction time on detection system: prepare 0, 10ppb, 50ppb, 100ppb cyromazine solutions respectively, and set the excitation wavelength to 480nm and use fluorescence spectrophotometry during the reaction time of 0 to 360 seconds with the detection solution. The fluorescence intensity at 520 nm was measured by a meter. The fluorescence intensity measured in the presence of cyromazine in the detection system is denoted as F, and the fluorescence intensity measured in the absence of cyromazine is denoted as F ₀ , and the quenching rate (F ₀ -F)/F ₀ is calculated. Figure 4 shows the reaction time and the influence on the detection system. It can be seen from the figure that the optimal reaction time of the system is after 2 minutes.

(5) Selectivity test of detection system: 15ppb cyromazine was added to milk samples, and 50ppb of common residual pollutants in milk were oxytetracycline, thiamphenicol, chloramphenicol, amitraz, and chlorhydrin. Pyridine, abamectin, levamisole, oxytetracycline, set the excitation wavelength to 480nm, and measure their fluorescence intensity at 520nm with a spectrofluorometer. The fluorescence intensity measured by the presence of veterinary drugs in the detection system was recorded as F, and the fluorescence intensity measured by blank was recorded as F ₀ , and the quenching rate (F ₀ -F)/F ₀ was calculated. Fig. 5 is a graph showing the results of the determination of the selectivity of the detection system. It can be seen from the figure that the detection system has good selectivity.

Example 2

(1) Preparation of detection solution: connect fluorescein FAM to the 5' end of the ssDNA whose sequence is GGTTGGTTGGTTGGTTTT to obtain the final cyromazine aptamer 5'-FAM-GGTTGGTTGGTTGGTTTT-3'. The aptamer content was diluted to a concentration of 25 nM with Tris-acetic acid buffer (10 mM, pH 8.0) to obtain a detection solution.

(2) Pretreatment of milk samples: In a 10 mL centrifuge tube, add 5 mL of 1% acetic acid and 1.0 mL of milk samples, and let stand for 5 minutes. Then centrifuge at 10,000 r for 10 minutes, and after taking the supernatant, adjust the pH to 8.0 with 2.0 mol/L NaOH.

(3) prepare the detection system of the known cyromazine concentration: get 10 centrifuge tubes containing the detection solution, add the milk samples of the cyromazine standard solutions of different concentrations after the pretreatment respectively, so that the ring in the whole detection system is The content of imiazine was maintained at 0-10ppb, and was measured after reacting at 25°C for 2min.

(4) Take the prepared standard solution, place it in a cuvette, set the excitation wavelength to 480 nm, and measure its fluorescence intensity at 520 nm with a fluorescence spectrophotometer. The fluorescence intensity measured in the presence of cyromazine in the detection system is denoted as F, and the fluorescence intensity measured in the absence of cyromazine is denoted as F ₀ , and the quenching rate (F ₀ -F)/F ₀ is calculated.

(5) Draw a standard curve by plotting different concentrations of cyromazine and the corresponding quenching rate (F ₀ -F)/F ₀ . Figure 6 is a standard curve diagram of cyromazine concentration and fluorescence quenching rate (F ₀ -F)/F ₀ , wherein the cyromazine concentrations are 0, 1, 2, 3, 4, 5, 6, 7, respectively , 8, 9, 10, 15, 20, 40, 60, 80, 100 and 200ppb, the Tcy2 concentration was 25nM.

(6) Preparation of the sample system to be tested: after pretreatment, mix with the detection solution, fully mix and react for 2 minutes, and measure its quenching rate (F ₀ -F)/F ₀ according to step (4).

(7) According to the quenching rate (F ₀ -F)/F ₀ obtained by the sample, and checking the standard curve, the content of cyromazine in the sample can be obtained.

(8) Verification: Four milks containing cyromazine with concentrations of 0.1, 0.2, 0.5, and 1.0 ppm were measured by the method of the present invention, and the recoveries obtained were 120%, 95%, 106%, and 106%, respectively, proving that reliability of this method.

(9) The minimum detection limit of the method of the present invention for measuring cyromazine in milk is 0.68ppb.

Claims (3)

1. a method for detecting cyromazine in milk based on G-quadruplex nucleic acid aptamer fluorescent probe, is characterized in that, concrete steps are as follows: (1) at the temperature of 24～26 ℃, after mixing 2min, obtain that the cyromazine content is maintained at A standard solution between 0-10ppb; wherein: the detection solution is obtained by diluting a FAM-modified nucleic acid aptamer with Tris-acetic acid buffer, and the FAM-modified nucleic acid aptamer is 5'-FAM-GGTTGGTTGGTTGGTTTT-3 ; (2) get the standard solution prepared above, place in the cuvette, after setting the excitation wavelength to be 480nm, measure its fluorescence intensity at 520nm with a fluorescence spectrophotometer; there is the fluorescence that cyromazine measures in the detection system The intensity is denoted as F, the fluorescence intensity measured in the absence of cyromazine is denoted as F ₀ , and the quenching rate (F ₀ -F)/F ₀ is calculated; (3) cyromazine of different concentrations and corresponding quenching rate (F ₀ -F)/F ₀ are plotted, and standard curve is drawn; (4) After pre-treatment of the milk sample to be detected, mix it with the detection solution of the same concentration in step (1), fully mix and react for 1-3 minutes, and use a fluorescence spectrophotometer to test the mixture at an excitation wavelength of 480 nm at 520 nm. The fluorescence intensity of , and then calculate its quenching rate (F ₀ -F)/F ₀ ; (5) According to the quenching rate (F ₀ -F)/F ₀ of the milk sample to be detected, check the standard curve to obtain the content of cyromazine in the milk sample to be detected. 2 . The method according to claim 1 , wherein the concentration of the detection solution is 20-30 nmol/L. 3 . 3. method according to claim 1, is characterized in that, the step of pretreatment of milk sample is as follows: first mix the 1wt% acetic acid of 4～6mL and the milk sample of 1.0mL, let stand for 5 minutes; Then 8000～12000r /min centrifugation for 8-12 minutes, after taking the supernatant, adjust the pH to 8.0 with 1.5-2.5mol/L NaOH.

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