CN103994984A - Method for performing colorimetric detection on tetracycline by aptamer and positive electricity AuNPs - Google Patents
Method for performing colorimetric detection on tetracycline by aptamer and positive electricity AuNPs Download PDFInfo
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- CN103994984A CN103994984A CN201410245653.6A CN201410245653A CN103994984A CN 103994984 A CN103994984 A CN 103994984A CN 201410245653 A CN201410245653 A CN 201410245653A CN 103994984 A CN103994984 A CN 103994984A
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- 239000004098 Tetracycline Substances 0.000 title claims abstract description 49
- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 49
- 229960002180 tetracycline Drugs 0.000 title claims abstract description 48
- 229930101283 tetracycline Natural products 0.000 title claims abstract description 48
- 150000003522 tetracyclines Chemical class 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 19
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 title claims description 11
- 230000005611 electricity Effects 0.000 title description 8
- 238000010521 absorption reaction Methods 0.000 claims abstract description 9
- 235000013305 food Nutrition 0.000 claims abstract description 6
- 238000004220 aggregation Methods 0.000 claims abstract 4
- 230000002776 aggregation Effects 0.000 claims abstract 4
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 claims abstract 3
- 229960003151 mercaptamine Drugs 0.000 claims abstract 3
- 239000000243 solution Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000004737 colorimetric analysis Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 2
- OGMADIBCHLQMIP-UHFFFAOYSA-N 2-aminoethanethiol;hydron;chloride Chemical compound Cl.NCCS OGMADIBCHLQMIP-UHFFFAOYSA-N 0.000 claims 1
- 229940097265 cysteamine hydrochloride Drugs 0.000 claims 1
- 239000012528 membrane Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000003814 drug Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 description 2
- 229940074095 ractopamine Drugs 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 208000031295 Animal disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229930195503 Fortimicin Natural products 0.000 description 1
- 229910004042 HAuCl4 Inorganic materials 0.000 description 1
- 231100000703 Maximum Residue Limit Toxicity 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- KIPLYOUQVMMOHB-MXWBXKMOSA-L [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O Chemical compound [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O KIPLYOUQVMMOHB-MXWBXKMOSA-L 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- BIDUPMYXGFNAEJ-APGVDKLISA-N astromicin Chemical compound O[C@@H]1[C@H](N(C)C(=O)CN)[C@@H](OC)[C@@H](O)[C@H](N)[C@H]1O[C@@H]1[C@H](N)CC[C@@H]([C@H](C)N)O1 BIDUPMYXGFNAEJ-APGVDKLISA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 description 1
- 229960004475 chlortetracycline Drugs 0.000 description 1
- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 description 1
- 235000019365 chlortetracycline Nutrition 0.000 description 1
- 210000003298 dental enamel Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940063650 terramycin Drugs 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
一种利用适配体和正电AuNPs比色检测四环素的方法，属于分析化学技术领域；其特征在于利用适配体可以引起巯基乙胺修饰的AuNPs的聚集，从而使AuNPs溶液的颜色由酒红色变为蓝色，吸收峰发生红移，四环素与适配体先作用后形成四环素-适配体结合物后不会引起AuNPs聚集，可以通过肉眼和紫外分光光度计检测食品中的四环素的残留量，该方法可简单、快速、灵敏的检测四环素，为以后的研究、生产、监管等提供了方便。
A method for colorimetrically detecting tetracycline by using an aptamer and positively charged AuNPs belongs to the technical field of analytical chemistry; it is characterized in that the use of aptamers can cause the aggregation of AuNPs modified by mercaptoethylamine, so that the color of the AuNPs solution changes from wine red to It is blue, and the absorption peak is red-shifted. Tetracycline interacts with the aptamer first and then forms a tetracycline-aptamer conjugate that will not cause aggregation of AuNPs. The residual amount of tetracycline in food can be detected by naked eyes and ultraviolet spectrophotometer. The method can detect tetracycline simply, rapidly and sensitively, and provides convenience for future research, production, supervision and the like.
Description
Technical field
The method of utilizing aptamers and positive electricity AuNPs colorimetric detection tetracycline, belongs to technical field of analytical chemistry.
Background technology
Tetracyclines is broad spectrum antibiotic, is a class the most cheap in microbiotic, and conventional tetracycline medication has tetracycline, aureomycin, terramycin, fortimicin etc.
In tetracycline medication, the use of tetracycline is comparatively extensive, cheap price makes tetracycline popular in developing country, not only be widely used in the treatment of the mankind and Animal diseases as medicine, also use in a large number as adjuvant simultaneously, for prevention and treatment livestock and poultry, be used for improving food utilization efficiency, promote growth of animal.But because the use of such medicine is unreasonable and abuse, easily induction produces antibody-resistant bacterium, also can cause occurring in the residue problem of tetracycline in food and milk.European Union and China all in regulation milk the maximum residue limit of tetracycline be 0.1mg/kg.
Child on long-term Reusability tetracycline, likely affects growth and the formation of tooth, not only makes tooth flavescence, also can cause enamel depauperation (dental surface is rough, occurs dolly dimple) or deformity teeth.Also easily suffer from carious tooth simultaneously.Gerontal patient's overdose, often with symptoms such as renal hypofunctions, has a strong impact on health, brings major hidden danger to human health.
The residual assay method of tetracycline mainly contains microbial method, euzymelinked immunosorbent assay (ELISA) (ELISA), thin-layered chromatography and liquid phase chromatography (HPLC), liquid chromatography-mass spectrography (LC-MS) etc., although these methods are highly sensitive, but there is the shortcoming of poor specificity in these methods, these methods need expensive equipment, minute is longer, cost is high, needs professional and technical personnel to operate, and is not suitable for field quick detection.Therefore need to set up a kind of quick, simple, sensitive method and detect the tetracycline in food.
Summary of the invention
The object of this invention is to provide the method for utilizing aptamers and positive electricity AuNPs colorimetric detection tetracycline, with the residual quantity of antibiotic tetracycline in quick, simple, sensitive detection food.
Technical matters: the method for utilizing aptamers and positive electricity AuNPs colorimetric detection tetracycline is based on following principle:
Freshly prepd positive electricity AuNPs, the AuNPs that mercaptoethylmaine is modified is uniformly dispersed, and because the effect of AuNPs surface amino groups is and positively charged, solution presents claret.
When adding after aptamers, because it contains the phosphate radical with negative electricity, because making AuNPs, electrostatic adsorption assembles, and color becomes blueness by claret, absorption peak generation red shift.
If tetracycline and aptamers first act on after rear formation tetracycline-aptamers bond, joining in positive electricity AuNPs solution, just can not cause that AuNPs assembles, solution still presents claret.
Technical scheme of the present invention: the preparation of the AuNPs that mercaptoethylmaine is modified; Utilize colourimetry to observe and detect the system of the tetracycline that contains variable concentrations.
(1) preparation of the AuNPs that mercaptoethylmaine is modified
First, 400 Mercaptamines of μ L0.213M and the HAuCl of 40mL1.40mM
4mixed liquor join in the round-bottomed flask of 100mL.This mixed liquor stirs 20 minutes in the dark, adds fast the NaBH of the new configuration of 10 μ L10mM
4solution, continues to stir in the dark 30 minutes.Obtained solution is cooled to after room temperature and removes sediment with the microporous barrier of 0.4 μ m, and filtrate is placed in 4 DEG C of refrigerators and uses in order to detecting.
(2) utilize colourimetry to observe and detect the system of the tetracycline that contains variable concentrations
Prepare following mixed solution: respectively the 40 tetracycline standard items of μ L variable concentrations and the aptamers of 20 μ L50nM are joined in the centrifuge tube of 1.5mL, vortex concussion 14min, then add the AuNPs of 40 μ L, final volume is diluted with water to 500 μ L, under room temperature, react 12min.The color of AuNPs and absorption peak can change along with the variation of tetracycline concentration; According to A
522concentration Criterion curve with tetracycline.
Tetracycline standard items ultimate density used is followed successively by: 0.2 μ g/mL, 0.4 μ g/mL, 0.6 μ g/mL, 1 μ g/mL, 1.2 μ g/mL, 1.6 μ g/mL, 2 μ g/mL.The range of linearity is 0.2 μ g/mL-2 μ g/mL.
Beneficial effect of the present invention: the present invention has prepared the AuNPs that mercaptoethylmaine is modified, and has set up simple, quick, a sensitive signalling, can fast detecting tetracycline, for supervision from now on provides convenience.
Brief description of the drawings
The uv absorption figure of the different systems of Fig. 1: (a) AuNPs, (b) AuNPs – tetracycline, (c) AuNPs-tetracycline-aptamers, (d) AuNPs-aptamers AuNPs:8.3 × 10
-10m, tetracycline, 2 μ g/mL.
The tetracycline standard items of Fig. 2 variable concentrations and the uv absorption figure of AuNPs effect: a-g:0.2 μ g/mL, 0.4 μ g/mL, 0.6 μ g/mL, 1 μ g/mL, 1.2 μ g/mL, 1.6 μ g/mL, 2 μ g/mL.
The canonical plotting of Fig. 3 Ractopamine: with A
522concentration Criterion curve with tetracycline.
The affect figure of Fig. 4 differential responses time (2min-20min detects once for every two minutes) on AuNPs-tetracycline-aptamers system: tetracycline, 2 μ g/mL, aptamers, 2nM.
Embodiment
The preparation of the AuNPs that mercaptoethylmaine is modified
Material/agent: HAuCl
4purchase in Beijing chemical reagents corporation
Method: first, the mixed liquor of 400 Mercaptamines of μ L0.213M and the HAuCl4 of 40mL1.40mM joins in the round-bottomed flask of 100mL.This mixed liquor stirs 20 minutes in the dark, adds fast the NaBH of the new configuration of 10 μ L10mM
4solution, continues to stir in the dark 30 minutes.Obtained solution is cooled to after room temperature and removes sediment with the microporous barrier of 0.4 μ m, and filtrate is placed in 4 DEG C of refrigerators and uses in order to detecting.
The ultra-violet absorption spectrum of result: AuNPs has maximum absorption band at 527nm place, and this is consistent with bibliographical information.
Because the system of experiment is subject to the impact in reaction time, so need the impact of research system time variation.
Material/agent: the AuNPs that mercaptoethylmaine is modified; Tetracycline standard items are purchased from life work biology (Shanghai) incorporated company.
Method: the aptamers of 40 μ L25 μ g/mL tetracycline standard items and 20 μ L50nM is joined in the centrifuge tube of 1.5mL, vortex shakes the different time (2min-20min), after every two minutes, add the AuNPs of 40 μ L, final volume is diluted with water to 500 μ L, under room temperature, react 12min, measure ultra-violet absorption spectrum.
Result: description time is influential to whole system, in the time that the reaction time is 14 minutes, reacts the most complete, therefore, finally chooses the 14 minutes reaction time as this reaction.
The method is for the detection of Ractopamine
Material/agent: the AuNPs that mercaptoethylmaine is modified; Tetracycline standard items are purchased from life work biology (Shanghai) incorporated company.
Method: (1) joins the 40 tetracycline standard items of μ L variable concentrations and the aptamers of 20 μ L50nM in the centrifuge tube of 1.5mL respectively, vortex concussion 14min, then add the AuNPs of 40 μ L, final volume is diluted with water to 500 μ L, under room temperature, react 12min.The color of AuNPs and absorption peak can change along with the variation of tetracycline concentration;
(2) tetracycline standard items ultimate density used is followed successively by: 0.2 μ g/mL, 0.4 μ g/mL, 0.6 μ g/mL, 1 μ g/mL, 1.2 μ g/mL, 1.6 μ g/mL, 2 μ g/mL.
Result: according to A
522concentration Criterion curve with tetracycline; The range of linearity is 0.2 μ g/mL-2 μ g/mL.
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Cited By (6)
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| 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 |
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| CN106645129A (en) * | 2017-01-03 | 2017-05-10 | 吉林大学 | Method for detecting chlorpyrifos through functionalized gold nanoparticle based novel colorimetric sensor |
| CN113281507A (en) * | 2021-05-23 | 2021-08-20 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
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- 2014-06-05 CN CN201410245653.6A patent/CN103994984A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104897596A (en) * | 2015-05-25 | 2015-09-09 | 东华大学 | Method for detecting chiral compound based on aptamer modified nanogold |
| 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 |
| CN106645129A (en) * | 2017-01-03 | 2017-05-10 | 吉林大学 | Method for detecting chlorpyrifos through functionalized gold nanoparticle based novel colorimetric sensor |
| CN113281507A (en) * | 2021-05-23 | 2021-08-20 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
| CN114778461A (en) * | 2022-04-22 | 2022-07-22 | 南通大学 | Preparation method of intelligent gold nanoparticles and application of intelligent gold nanoparticles in beta-amyloid protein colorimetric analysis |
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