CN101701260B - Method for detecting melamine based on oligonucleotide-non-labeled nano-Au - Google Patents
Method for detecting melamine based on oligonucleotide-non-labeled nano-Au Download PDFInfo
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- CN101701260B CN101701260B CN2009102724577A CN200910272457A CN101701260B CN 101701260 B CN101701260 B CN 101701260B CN 2009102724577 A CN2009102724577 A CN 2009102724577A CN 200910272457 A CN200910272457 A CN 200910272457A CN 101701260 B CN101701260 B CN 101701260B
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- oligonucleotide
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- trimeric cyanamide
- buffered soln
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Abstract
The invention discloses a method for detecting melamine based on oligonucleotide-non-labeled nano-Au, comprising the following steps of: 1) preparing nano-Au colloidal sol with certain grain diameter, namely, reaction liquid A; 2) dissolving oligonucleotide solution with special sequence into buffer solution, and obtaining reaction liquid B; 3) adding solution to be detected into the reaction liquid B for reaction; and 4) adding the reaction liquid A into the reaction liquid in the step 3), and detecting the melamine by colorimetry. The detection method is simple, convenient and rapid (the detection process is not longer than 10min), high in sensitivity, good in selectivity and low in cost, can reach the detection requirement in provisional management limited quantity regulation of melamine in milk and dairy products of China without needing large-scale instruments and equipment, and can realize field rapid detection for raw materials and products.
Description
Technical field
The present invention relates to the interdisciplinary field of subjects such as biology, medical science, bromatology, chemistry.More specifically relate to a kind of method that detects trimeric cyanamide based on oligonucleotide-non-labeled nanometer gold.
Background technology
Trimeric cyanamide (Melamine) has caused people's extensive concern to the harm that humans and animals causes; The false albumen incident of liquid state milk and milk powder mainly is because some lawless people are added trimeric cyanamide in milk or milk powder; Improve the nitrogen content of milk preparation, pretend to be protein with this.After yet trimeric cyanamide gets into human body, substitution reaction (hydrolysis) takes place, generate tricyanic acid, tricyanic acid and trimeric cyanamide form big reticulated structure, and cause calculus with urine formation indissoluble salt, and health is constituted a serious threat.On October 7th, 2008, the Ministry of Health, Ministry of Industry and Information, the Ministry of Agriculture, State Administration for Industry & Commerce and State General Administration for Quality Supervision united issue " about the bulletin of the trimeric cyanamide temporary control and education value of limiting the quantity of regulation in breast and the milk-product ", and the bulletin regulation: the value of limiting the quantity of of trimeric cyanamide is 1mg/kg in the baby formula milk powder; The value of limiting the quantity of of trimeric cyanamide is 2.5mg/kg in liquid state milk (comprising raw dairy), milk powder, other prescription emulsifiable powders.The melamine detection method mainly contains at present: liquid phase chromatography (HPLC), LC/MS (HPLC-MS), gas chromatography-mass spectrography (GC-MS) etc.But above-mentioned detection method all need be used large-scale instrument, and complex operation is wasted time and energy, and cost is high, can't directly carry out rapid detection to trimeric cyanamide at raw material and product scene.
The dispersion of nm gold particles and aggregating state have obvious color to change, and this phenomenon has been widely used in the colourimetry detection of protein, DNA etc. in recent years, are that the method for probe in detecting trimeric cyanamide is based upon on the basis of this principle just with the nanometer gold.Though not long ago the someone utilized this principle to report a similar detection method (Kelong Ai, Yanlan Liu, and Lehui Lu; Journal of the American ChemicalSociety 2009,131 (27), 9496-9497.); But their reported method not only need be used SR marking nano gold, and this reagent do not have commercialization, needs own synthesizing; Not only labour intensity is big, waste time and energy, and its purity directly influences detection sensitivity and selectivity.
Summary of the invention
The objective of the invention is to be to provide a kind of method that detects trimeric cyanamide based on oligonucleotide-non-labeled nanometer gold; With the nanometer gold is the probe in detecting trimeric cyanamide, easy to be quick, highly sensitive; Selectivity is good; Cost is low, need not large-scale instrument and equipment and can reach the requirement of country about the trimeric cyanamide temporary control and education value of limiting the quantity of regulation in breast and the milk-product, can realize the rapid detection of trimeric cyanamide in raw material and the product.
To achieve these goals, the present invention adopts following technical measures:
A kind of method based on oligonucleotide-non-labeled nanometer gold detection trimeric cyanamide, its step is following:
1) nanometer gold is synthetic: with hydrochloro-auric acid (HAuCl
44H
2O), trisodium citrate, ultrapure water be raw material, through magnetic agitation, heating (100 ℃), backflow, cooling operation technology, the preparation particle diameter is that 10nm～40nm, concentration are 10
-11M～10
-8M reaction solution A, described reaction solution A is a nano gold sol, and the mol ratio of described hydrochloro-auric acid and trisodium citrate is 1: 1～1: 5, and preferable temperature of reaction is 80 ℃～100 ℃, and the reaction times is 25～60min.
2) the particular sequence oligonucleotide is dissolved in the buffered soln, gets reaction solution B.Described particular sequence oligonucleotide is single strain oligonucleotide (worker is given birth in Shanghai), and base wherein is the T base or contains the oligonucleotide of different number T bases, and the base number is 1～200, and its concentration is 10
-6M～10
-4M; Described buffered soln is conventional Tris, PBS buffered soln, and its concentration is 5～20mM; Contain sodium-chlor in the described buffered soln, its concentration is 0.05～0.5M.
3) in reaction solution B, add trimeric cyanamide or the pretreated sample of process, fully mixing leaves standstill.Described reaction solution B is a step 2) in the buffered soln of particular sequence oligonucleotide; Described pretreated sample is the pretreated sample of process standard method; Described melamine concentration is 10
-9M～10
-6M; Time of repose is 1～10min.
4) with reaction solution A as probe, through colourimetry, can detect trimeric cyanamide.Described reaction solution A is the nano gold sol in the step 1), and the preferable mol ratio of described reaction solution A and particular sequence oligonucleotide (worker is given birth in Shanghai) is 1: 100～1: 1000; Described colourimetry is visual colorimetry or visible spectrophotometry, and it is 400～750nm that visible spectrophotometry detects wavelength.
This detection method is used for sample melamine detection such as raw material milk, liquid milk product, milk powder, feed, can in 10 minutes, accomplish.
The present invention compared with prior art has the following advantages and effect:
With liquid phase chromatography (HPLC), LC/MS (HPLC-MS), gas chromatography-mass spectrography compared with techniques such as (GC-MS), need not large-scale instrument and equipment, the testing staff need not special training, only gets final product judged result with bore hole; Can be used for on-the-spot the detection; Directly adopt commercial oligonucleotide; Can realize the colorimetric detection to trimeric cyanamide, its detection sensitivity can reach national standard, have easy quick, highly sensitive, selectivity is good, cost is low; Need not large-scale instrument, can carry out the scene detection of trimeric cyanamide raw material and product.
This method detects (in 10 minutes) fast, operates simple and easyly, highly sensitive, and selectivity is good, highly versatile.
Description of drawings
Fig. 1 is a probe with cold nanometer gold, detects the trimeric cyanamide principle schematic in conjunction with oligonucleotide
Fig. 2 base T and trimeric cyanamide form the hydrogen bond synoptic diagram
Fig. 3 A synthetic nano gold sol Electronic Speculum figure
Fig. 3 B is a probe with reaction solution A, has just begun the nanometer gold Electronic Speculum figure that reunites behind the adding trimeric cyanamide
Fig. 3 C is a probe with reaction solution A, adds the nanometer gold Electronic Speculum figure that reunites behind the excessive slightly trimeric cyanamide
The oligonucleotide-non-labeled nanometer gold of Fig. 4 A detects the UV spectrum and the linear relationship chart of trimeric cyanamide
The oligonucleotide-non-labeled nanometer gold of Fig. 4 B detects trimeric cyanamide visual colorimetry synoptic diagram
Embodiment
Embodiment 1:
With melamine detection in the raw material milk is that example specifies detection method, and its step is following:
1) the used instrument of synthesis of nano gold all need use new preparation chloroazotic acid (1: 3, V
Concentrated nitric acid/ V
Concentrated hydrochloric acid) soak 24h after, ultrapure water is cleaned subsequent use.
2) with 100mL ultrapure water heated and boiled, (2%, w/w), the boiling back adds 10.0mL trisodium citrate (the trisodium citrate quality is 0.1141g) under heating (100 ℃) and agitation condition to add the 1.88mL hydrochloro-auric acid.10.0 stop heating after minute, continue to stir 15.0 minutes, naturally cool to room temperature (20-25 ℃).The mol ratio of described hydrochloro-auric acid and trisodium citrate is 1: 3.88.Being prepared into the nano gold sol particle diameter is that 13nm, concentration are 4.86 * 10
-9M.
3) the particular sequence oligonucleotide is dissolved in the buffered soln, gets reaction solution B.Described particular sequence oligonucleotide is a single strain oligonucleotide, and base wherein is the T base or contains the more oligonucleotide of T base, and its concentration is 10
-5M; Described buffered soln is PBS buffered soln, and its concentration is 10mM; Sodium chloride-containing in the described buffered soln, its concentration are 0.3M.
4) in reaction solution B, add through pretreated raw material milk sample, fully mixing leaves standstill.Described reaction solution B is the particular sequence oligonucleotide buffered soln in the step 3); Described pretreated raw material milk sample must be earlier through trichoroacetic acid(TCA) or acetonitrile precipitation protein, then through the pretreated sample of self-control SPE integral post; Time of repose is 5min.
5) with reaction solution A as probe, through colourimetry, can detect trimeric cyanamide in the raw material milk.Described reaction solution A is a step 2) in nano gold sol, the preferable mol ratio of described reaction solution A and particular sequence oligonucleotide is 1: 100; Described colourimetry is visual colorimetry or visible spectrophotometry, and visible spectrophotometry detects wavelength region: 400～700nm.
The result:
Characterize the concentration change of trimeric cyanamide with the absorbance at 520nm place, its linearity range is: 4.17 * 10
-8～4.17 * 10
-7M detects and is limited to 41.7nM.
Claims (1)
1. the method based on oligonucleotide-non-labeled nanometer gold detection trimeric cyanamide the steps include:
1) nanometer gold is synthetic: with hydrochloro-auric acid, trisodium citrate, ultrapure water is raw material, and through magnetic agitation, 100 ℃ of heating, backflow, cooling operation, the preparation particle diameter is that 10nm～40nm, concentration are 10
-11M～10
-8M reaction solution A, described reaction solution A is a nano gold sol, and the mol ratio of described hydrochloro-auric acid and trisodium citrate is 1: 1～1: 5, and the reaction times is 25～60min;
2) the particular sequence oligonucleotide is dissolved in the buffered soln; Get reaction solution B, described particular sequence oligonucleotide is a single strain oligonucleotide, and the base number is 2～200; Base wherein is the T base or contains the oligonucleotide of different number T bases, and its concentration is 10
-6M～10
-4M; Described buffered soln is Tris or PBS buffered soln, and its concentration is 5～20mM; Contain sodium-chlor in the described buffered soln, its concentration is 0.05～0.5M;
3) in reaction solution B, add trimeric cyanamide standardized solution or through pretreated sample, mixing leaves standstill, described reaction solution B is a step 2) in the buffered soln of particular sequence oligonucleotide; Described pretreated sample is the pretreated sample of process standard method; Described melamine concentration is 10
-9M～10
-6M; Time of repose is 1～10min;
4) with reaction solution A as probe; Through visual colorimetry or ultraviolet visible spectrophotometry; Detect trimeric cyanamide, described reaction solution A is the nano gold sol in the step 1), and the mol ratio of described reaction solution A and particular sequence oligonucleotide is 1: 100～1: 1000; It is 400～750nm that ultraviolet visible spectrophotometry detects wavelength.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101846631B (en) * | 2010-06-04 | 2011-10-05 | 江南大学 | Method for quickly testing melamine content in milk powder by using gold nanoparticle colorimetric method |
| CN102608073A (en) * | 2012-03-09 | 2012-07-25 | 西南大学 | Quick detection method for melamine in dairy products |
| CN102749301B (en) * | 2012-06-27 | 2014-06-18 | 浙江大学宁波理工学院 | Method for determining melamine content by using ultraviolet spectroscopy |
| CN102768207B (en) * | 2012-08-01 | 2014-10-29 | 福建医科大学 | Method for detecting tripolycyanamide based on nanogold mimetic peroxidase |
| CN103163101B (en) * | 2013-03-14 | 2016-06-15 | 四川大学 | A kind of method of trimeric cyanamide in quick mensuration milk preparation |
| CN103364352B (en) * | 2013-07-19 | 2015-04-08 | 福建医科大学 | Method for rapid determination of sulphur ions with nanogold as logic gate color developing probe |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182580A (en) * | 2007-11-19 | 2008-05-21 | 中国科学院上海微系统与信息技术研究所 | Gene or gene mutation measuring method based on magnetic beads and nanometer gold detecting probe |
| CN101424642A (en) * | 2008-11-14 | 2009-05-06 | 中国科学院上海应用物理研究所 | Target molecule detecting method based on nanometer aurum and nucleic acid structure |
| CN101539578A (en) * | 2009-02-17 | 2009-09-23 | 李红玉 | Colloidal gold test strip for testing melamine content |
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2009
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182580A (en) * | 2007-11-19 | 2008-05-21 | 中国科学院上海微系统与信息技术研究所 | Gene or gene mutation measuring method based on magnetic beads and nanometer gold detecting probe |
| CN101424642A (en) * | 2008-11-14 | 2009-05-06 | 中国科学院上海应用物理研究所 | Target molecule detecting method based on nanometer aurum and nucleic acid structure |
| CN101539578A (en) * | 2009-02-17 | 2009-09-23 | 李红玉 | Colloidal gold test strip for testing melamine content |
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