CN103149167A - Method for detecting tetracycline residues in milk and drinking water - Google Patents
Method for detecting tetracycline residues in milk and drinking water Download PDFInfo
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- CN103149167A CN103149167A CN2013100594325A CN201310059432A CN103149167A CN 103149167 A CN103149167 A CN 103149167A CN 2013100594325 A CN2013100594325 A CN 2013100594325A CN 201310059432 A CN201310059432 A CN 201310059432A CN 103149167 A CN103149167 A CN 103149167A
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Abstract
The invention discloses a method for detecting tetracycline residues in milk and drinking water and belongs to the technical field of food safety. The method comprises the following steps of: constructing a detection system with known tetracycline concentration to obtain a light absorption difference standard curve, fitting the curve to obtain a regression equation, mixing milk or drinking water to be detected with an acetic acid solution to obtain a mixed solution, centrifuging and layering the mixed solution to obtain a supernatant liquid, adding a tetracycline aptamer solution, adding a nanogold solution after incubation, finally adding positive ion electrolyte, determining the difference of the light adsorption rates at the 650nm part and the 520nm part of an electrolyte standard solution and a blank control solution by a microplate reader, and calculating the content of the tetracycline in milk and drinking water to be detected under the control of the equation obtained by fitting the standard curve. The method is high in speed, low in cost, high in sensitivity, good in selectivity, and convenient to operate without depending on large-sized instruments or equipment, the lowest detection limit is 45.8nM, the defects that the existing aptamer is time-wasting due to marking and high in cost are overcome and the method has the advantages of high speed, sensitivity and low cost.
Description
Technical field
What the present invention relates to is a kind of method of food security technical field, specifically a kind ofly utilizes cationic polyelectrolyte to assemble nanogold particle to detect that in milk and potable water, the tetracycline concentration range is 1-5 μ M, and lowest detection is limited to the method for 45.8nM.
Background technology
Tetracycline is that a class is controlled the broad-spectrum antibiotic that bacterium infects and Profilin is synthetic.In view of its good biocidal property and low price, tetracycline is widely used in humans and animals treatment, growth promotion and makes an addition in animal and fowl fodder.But the excessive use energy inducible strain drug resistance of tetracycline, and cause high medicament residue, this will produce direct or indirect harm to human health.In order to guarantee consumer's food security, nineteen ninety, in European Union's regulation milk, the maximum residue limit of tetracycline is 225nM; In U.S. FDA regulation milk, the tetracycline maximum residue limit is 900nM.2002, in regulation ox/goat milk and all animal foods, the high residue amount of tetracycline medication was 100 μ g/kg in " the animal food herbal medicine maximum residue limit(MRL) " of China Ministry of Agriculture revision.
The tetracycline detection method of now having reported is a lot, as instrument analytical approach (high performance liquid chromatography (HPLC), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), fluorescence analysis), Antimicrobial method and immunization etc.Instrument analytical method needs high-end analytical equipment and professional person's operation, and time-consuming very long, can not satisfy fast, efficiently and immediately detect.The Antimicrobial method is a kind of comparatively conventional microbiotic detection method, but its poor specificity, time and effort consuming, measuring result error is also very large.Immunization is the many a kind of detection methods of present research and comparison, have the characteristics such as high specificity, sensitivity height, but its testing process is quite loaded down with trivial details, needs preparation plurality of antigens antibody, and other laboratory equipment such as microplate reader etc., analysis time, length needed about 4 hours.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of residual method of tetracycline in milk and potable water that detects is proposed, compete in conjunction with the tetracycline aptamers by cationic polyelectrolyte and tetracycline, form respectively supramolecular structure and tetracycline-aptamers compound, cause that in system, the nanogold particle aggregation extent changes and change color, and the system color becomes positive correlation with the colorimetric signal intensity with the tetracycline concentration that adds.Therefore this method is quick, cheap, highly sensitive, selectivity good, easy and simple to handle and do not need to rely on large-scale instrument and equipment, lowest detection is limited to 45.8nM, and having overcome in the past aptamers needs mark and time-consuming high in cost of production defective, has advantages of fast, sensitive and cheap.
The present invention realizes by following skill scheme:
The present invention relates to a kind of implementation method of tetracycline photosensitivity, by tetracycline is mixed with tetracycline aptamers solution, nano-Au solution and cationic polyelectrolyte successively, make mixed solution be directly proportional to tetracycline concentration at the light absorption ratio at 520nm and 650nm place.
The concentration range of described tetracycline is: 0.01-5 μ M
Described tetracycline aptamers solution prepares in the following manner: take 0.222g tetracycline pulvis and be dissolved in the 500mL volumetric flask, configuration concentration is 0.5mM tetracycline mother liquor, and gradient dilution obtains required tetracycline.
Described nano-Au solution refers to: the hypochlorous acid of 3.5mL1.0wt% is added in citric acid three sodium solutions of 10.5mL1.0%, add thermal agitation after 30 minutes, do not heat and be stirred to solution colour and become shiny red by grey, be set to the nano-Au solution that nanometer particle size is 15nm, be saved to 4 ° of C refrigerators.
Described cationic polyelectrolyte refers to: concentration is 0.1121nM polydiene the third dimethyl amine (PDDA) solution
the present invention relates to a kind of residual method of tetracycline in milk and potable water that detects, contain the detection system of known tetracycline concentration by structure after, obtain light absorption difference typical curve and match and obtain regression equation, then after milk to be measured or potable water being mixed with acetic acid solution, centrifugal layering obtains supernatant, add again tetracycline aptamers solution, add nano-Au solution after hatching, add at last cationic polyelectrolyte and measure the poor of the 650nm of tetracycline standard solution and blank liquid (replacing tetracycline in sample with ultrapure water) and 520nm place's light absorption ratio with microplate reader, the equation that the reference standard curve obtains calculates the tetracycline content in milk to be measured or potable water.
Described cationic polyelectrolyte is polydiene the third dimethyl amine (PDDA), and final concentration is 0.1121nM.
The DNA sequence dna of described tetracycline aptamers is:
5'-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGTCCCACTG CGCGTGGATCCGAGCTCCACGTG-3', the final concentration of this tetracycline aptamers solution is 6nM.
In described nano-Au solution, the particle diameter of nm of gold is 15nm.
The described detection system that contains known tetracycline concentration prepares in the following manner:
1) get 9 PE pipes that comprise tetracycline aptamers and buffer solution, add respectively 10 μ L variable concentrations tetracycline titers, make the tetracycline content in whole detection system maintain 0.01-5 μ M, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 15min;
2) separately get 1 PE pipe that comprises tetracycline aptamers and buffer solution, add 10 μ L ultrapure waters, by step 1) process afterwards as the blank system solution;
3) add respectively 100 μ L nano-Au solutions in 10 PE pipes, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 15min;
4) add respectively again the PDDA of 10 μ L concentration 55.5nM in above-mentioned 10 PE pipes of having hatched, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 5min.
Described light absorption difference typical curve refers to: with variable concentrations tetracycline (C
TET) as horizontal ordinate, measure the difference of ratio of the 650nm that obtains and 520nm place absorbance value with microplate reader take detection system and blank liquid as ordinate drawing standard curve.
The concentration of described acetic acid solution is 1%wt.;
Described mixing refers to: adopt the reaction of constant temperature oscillation instrument after 15 minutes, with the nylon ultrafiltration membrance filter of 150 μ m, use at last the ultrapure water constant volume to 500 μ L.
The equation that described typical curve match obtains is y=3.6386x+0.1414, and wherein: X is △ A=A650/A520, and with the substitution of X value, the y value of trying to achieve is the content of tetracycline in solution.
Principle of the present invention is: utilize cationic polyelectrolyte to have following two aspect characteristics: (1) positively charged kation can be combined by electrostatic interaction with electronegative aptamers phosphoric acid skeleton group; (2) cationic polyelectrolyte can destroy the charge balance between nanogold particle, impels nm of gold gathering and color to become blueness by redness.When not containing tetracycline in system, tetracycline aptamers and cationic polyelectrolyte interosculate, and consume the cationic polyelectrolyte in system, and the nanogold particle in solution is disperse state at this moment, and color is aobvious red.And when having tetracycline in system, tetracycline can preferentially be competed the tetracycline aptamers, and both form compound, and the cationic polyelectrolyte that exists in system can impel nanogold particle to assemble, and the system color becomes blueness by redness.Therefore system color and colorimetric signal intensity and the tetracycline significant positive correlation that adds by the method, can realize the detection of tetracycline by colorimetric analysis.
Technique effect
Compared with prior art, as long as qualitative detection of the present invention is by the visual inspection change color; Quantitatively detect detectability and can reach 45.8nM, well below European Union and domestic standard; Detect rapidly, can complete total Test in 2 hours, and the credible result degree is high; Adopt novel nucleic acids probe technology, opened up the detection technique frontier; Simple to operate, safety non-pollution, principle is applied widely.
Description of drawings
Fig. 1 is that the present invention detects schematic diagram.
Fig. 2 is aptamers concentration map in optimization method.
Fig. 3 is PDDA concentration map in optimization method.
Fig. 4 is tetracycline content and light absorption value graph of a relation.
Fig. 5 is the detection specificity figure of this method.
Embodiment
The below elaborates to embodiments of the invention, and the present embodiment is implemented under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
As shown in Figure 1, the present embodiment comprises the following steps:
Aptamers concentration in optimization system: adding respectively concentration range is that the 1-20nM aptamers is in nano-Au solution, add at last 0.1365 μ M PDDA, measure the difference curve plotting of the ratio of the 650nm obtain and 520nm place absorbance value with microplate reader, the aptamers concentration that peak value is corresponding is 6nM.
PDDA concentration in optimization system: with 6nM tetracycline aptamers respectively with 0.2,2 μ M tetracycline in hatch 15 minutes after, add 100 μ L nm of gold, adding at last concentration range is that the PDDA reaction of 0.084-0.158nM is after 5 minutes, measure the difference curve plotting of the ratio of the 650nm obtain and 520nm place absorbance value with microplate reader, the PDDA concentration that peak value is corresponding is 0.1121nM.
The tetracycline detection system of preparation concentration known: get 9 PE pipes that comprise tetracycline aptamer and nm of gold mixed liquor, hatched 15 minutes under 25 ℃ of conditions; Then add respectively 10 μ L variable concentrations tetracycline titers, make the tetracycline content in whole detection system maintain between 0.01 – 5 μ M, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 15min;
Separately get 1 PE pipe that comprises tetracycline aptamers and buffer solution, add 10 μ L ultrapure waters, by step 1) process afterwards as the blank system solution;
Add respectively 100 μ L nano-Au solutions in 10 PE pipes, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 15min;
Add respectively again the PDDA of 0.1121nM in above-mentioned 10 PE pipes of having hatched, fully PE is placed under 25 ° of C conditions again after mixing and hatches 5min.
Described light absorption difference typical curve refers to: with variable concentrations tetracycline (C
TET) as horizontal ordinate, measure the difference of ratio of the 650nm that obtains and 520nm place absorbance value with microplate reader take detection system and blank liquid as ordinate drawing standard curve.
After the dissolving of certain brand milk powder water, add acetic acid, get supernatant after centrifugal 3 times.Get 10 μ L supernatant samples and add 10 μ L tetracycline aptamers, react and added 10 μ L concentration 55.5 μ M PDDA in 15 minutes in backward test tube, hatch and add 100 μ L nm of gold after 10 minutes, add the pure water constant volume to 500 μ L standing 30 minutes.Get respectively and measure its light absorption value A under the condition that 200 μ L are 650nm and 520nm at the microplate reader wavelength
650,, A
520Ratio calculated, substitution formula y=3.6386x+0.1414 can try to achieve the y value.
Claims (10)
1. the implementation method of a tetracycline photosensitivity, it is characterized in that, by tetracycline is mixed with tetracycline aptamers solution, nano-Au solution and cationic polyelectrolyte successively, make mixed solution be directly proportional to tetracycline concentration at the light absorption ratio at 650nm and 520nm place.
2. method according to claim 1, is characterized in that, the concentration range of described tetracycline is: 0.01-5 μ M.
3. method according to claim 1, it is characterized in that, described tetracycline aptamers solution prepares in the following manner: take 0.222g tetracycline pulvis and be dissolved in the 500mL volumetric flask, configuration concentration is 0.5mM tetracycline mother liquor, and gradient dilution obtains required tetracycline.
4. method according to claim 1, it is characterized in that, described nano-Au solution refers to: the hypochlorous acid of 3.5mL1.0wt% is added in citric acid three sodium solutions of 10.5mL1.0%, add thermal agitation after 30 minutes, do not heat and be stirred to solution colour and become shiny red by grey, be set to the nano-Au solution that nanometer particle size is 15nm, be saved to 4 ° of C refrigerators.
5. method according to claim 1, is characterized in that, described cationic polyelectrolyte refers to: concentration is 0.1121nM polydiene the third dimethyl amine solution.
6. one kind is detected the residual method of tetracycline in milk and potable water, it is characterized in that, contain the detection system of known tetracycline concentration by structure after, obtain light absorption difference typical curve and match and obtain regression equation, then after milk to be measured or potable water being mixed with acetic acid solution, centrifugal layering obtains supernatant, add again tetracycline aptamers solution, add nano-Au solution after hatching, add at last cationic polyelectrolyte and measure the poor of the 650nm of tetracycline standard solution and blank liquid and 520nm place's light absorption ratio with microplate reader, the equation that the reference standard curve obtains calculates the tetracycline content in milk to be measured or potable water.
7. method according to claim 6, is characterized in that, described cationic polyelectrolyte is polydiene the third dimethyl amine, and final concentration is 0.1121nM;
The DNA sequence dna of described tetracycline aptamers is:
5'-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGTCCCACTG CGCGTGGATCCGAGCTCCACGTG-3', the final concentration of this tetracycline aptamers solution is 6nM;
In described nano-Au solution, the particle diameter of nm of gold is 15nm;
The concentration of described acetic acid solution is 1%wt.
8. method according to claim 6, is characterized in that, the described detection system that contains known tetracycline concentration prepares in the following manner:
1) get 9 PE pipes that comprise tetracycline aptamers and buffer solution, add respectively 10 μ L variable concentrations tetracycline titers, make the tetracycline content in whole detection system maintain 0.01-5 μ M, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 15min;
2) separately get 1 PE pipe that comprises tetracycline aptamers and buffer solution, add 10 μ L ultrapure waters, by step 1) process afterwards as the blank system solution;
3) add respectively 100 μ L nano-Au solutions in 10 PE pipes, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 15min;
4) add respectively again the PDDA of 10 μ L concentration 55.5nM in above-mentioned 10 PE pipes of having hatched, fully again the PE pipe is placed under 25 ° of C conditions after mixing and hatches 5min.
9. method according to claim 6, it is characterized in that, described light absorption difference typical curve refers to: as horizontal ordinate, measure the difference of ratio of the 650nm that obtains and 520nm place absorbance value with microplate reader take detection system and blank liquid as ordinate drawing standard curve with the variable concentrations tetracycline.
10. according to claim 6 or 9 described methods, is characterized in that, the equation that described typical curve match obtains is y=3.6386x+0.1414, and wherein: X is △ A=A650/A520, and with the substitution of X value, the y value of trying to achieve is the content of tetracycline in solution.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103335972A (en) * | 2013-07-16 | 2013-10-02 | 江南大学 | Detection method of kanamycin residue based on nucleic acid aptamer |
| CN103361353A (en) * | 2013-07-19 | 2013-10-23 | 暨南大学 | Tetracyclines aptamer and aptamer electrochemical biological sensor for detection of tetracyclines |
| CN104502585A (en) * | 2014-12-30 | 2015-04-08 | 中山大学 | Nano-sensor for detecting antibiotics and preparation method and application of nano-sensor |
| CN104597258A (en) * | 2015-01-09 | 2015-05-06 | 上海交通大学 | Method for detecting 17beta-estradiol by employing colorimetric method based on nucleic acid aptamer |
| CN104865216A (en) * | 2015-06-10 | 2015-08-26 | 常熟理工学院 | Method for colorimetrically detecting chloramphenicol based on nano-gold nucleic acid aptamer |
| CN104931467A (en) * | 2015-02-28 | 2015-09-23 | 青岛科技大学 | Intracellular adenosine triphosphate (ATP) fluorescence imaging method |
| US20160251660A1 (en) * | 2013-10-16 | 2016-09-01 | Korea University Research And Business Foundation | Nucleic acid aptamer capable of specifically binding to tebuconazole, mefenacet and inabenfide, and use therof |
| CN106290203A (en) * | 2016-10-17 | 2017-01-04 | 贵州大学 | A kind of tetracycline colorimetric detection method based on Catalysis by Hemin reaction |
| CN106706528A (en) * | 2016-11-24 | 2017-05-24 | 西安交通大学 | Aptamer-based tetracycline colorimetric detection method |
| CN109115705A (en) * | 2018-08-31 | 2019-01-01 | 上海应用技术大学 | A method of utilizing cyromazine in aptamer and PDDA assemble nanometer detection of particles milk |
| CN109799357A (en) * | 2019-01-09 | 2019-05-24 | 山西大学 | A kind of method of gold nanoparticle colorimetric detection antibiotic |
| CN110567952A (en) * | 2019-09-29 | 2019-12-13 | 上海应用技术大学 | aptamer-modified nanogold-based cyromazine detection method |
| CN113324933A (en) * | 2021-06-01 | 2021-08-31 | 浙江大学 | Method for detecting angiotensin converting enzyme and application thereof |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103335972A (en) * | 2013-07-16 | 2013-10-02 | 江南大学 | Detection method of kanamycin residue based on nucleic acid aptamer |
| CN103361353A (en) * | 2013-07-19 | 2013-10-23 | 暨南大学 | Tetracyclines aptamer and aptamer electrochemical biological sensor for detection of tetracyclines |
| US20160251660A1 (en) * | 2013-10-16 | 2016-09-01 | Korea University Research And Business Foundation | Nucleic acid aptamer capable of specifically binding to tebuconazole, mefenacet and inabenfide, and use therof |
| CN104502585A (en) * | 2014-12-30 | 2015-04-08 | 中山大学 | Nano-sensor for detecting antibiotics and preparation method and application of nano-sensor |
| CN104597258A (en) * | 2015-01-09 | 2015-05-06 | 上海交通大学 | Method for detecting 17beta-estradiol by employing colorimetric method based on nucleic acid aptamer |
| CN104931467A (en) * | 2015-02-28 | 2015-09-23 | 青岛科技大学 | Intracellular adenosine triphosphate (ATP) fluorescence imaging method |
| CN104931467B (en) * | 2015-02-28 | 2018-08-21 | 青岛科技大学 | A kind of fluorescence imaging method of intracellular ATP |
| CN104865216A (en) * | 2015-06-10 | 2015-08-26 | 常熟理工学院 | Method for colorimetrically detecting chloramphenicol based on nano-gold nucleic acid aptamer |
| CN106290203A (en) * | 2016-10-17 | 2017-01-04 | 贵州大学 | A kind of tetracycline colorimetric detection method based on Catalysis by Hemin reaction |
| CN106290203B (en) * | 2016-10-17 | 2019-04-16 | 贵州大学 | A kind of tetracycline colorimetric detection method based on Catalysis by Hemin reaction |
| CN106706528A (en) * | 2016-11-24 | 2017-05-24 | 西安交通大学 | Aptamer-based tetracycline colorimetric detection method |
| CN109115705A (en) * | 2018-08-31 | 2019-01-01 | 上海应用技术大学 | A method of utilizing cyromazine in aptamer and PDDA assemble nanometer detection of particles milk |
| CN109799357A (en) * | 2019-01-09 | 2019-05-24 | 山西大学 | A kind of method of gold nanoparticle colorimetric detection antibiotic |
| CN110567952A (en) * | 2019-09-29 | 2019-12-13 | 上海应用技术大学 | aptamer-modified nanogold-based cyromazine detection method |
| CN113324933A (en) * | 2021-06-01 | 2021-08-31 | 浙江大学 | Method for detecting angiotensin converting enzyme and application thereof |
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Application publication date: 20130612 |