CN101701259B - Method for detecting melamine based on oligonucleotides-modified nano-gold - Google Patents
Method for detecting melamine based on oligonucleotides-modified nano-gold Download PDFInfo
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- CN101701259B CN101701259B CN200910272456A CN200910272456A CN101701259B CN 101701259 B CN101701259 B CN 101701259B CN 200910272456 A CN200910272456 A CN 200910272456A CN 200910272456 A CN200910272456 A CN 200910272456A CN 101701259 B CN101701259 B CN 101701259B
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Abstract
The invention discloses a method for detecting melamine based on oligonucleotides-modified nano-gold, which comprises the steps of: 1) connecting the oligonucleotides which can be specifically combined with the melamine to the surface of the nano-gold to form a capture probe, modifying the nano-gold by adding the NaCl oligonucleotides into the nano-gold solution with a certain grain diameter, reaction temperature and reaction time as well as a certain concentration ratio of the nano-gold to the oligonucleotides, and introducing cysteine or glutathione during reacting; 2) reacting by adding the solution to be tested into the oligonucleotides-modified capture probe solution of the step 1), and combining the melamine with the oligonucleotides by adding the NaCl at a certain reaction concentration, reaction temperature and reaction time of the solution to be tested; and 3) cooling and diluting the reaction solution of the step 2), and detecting the melamine by means of visual colorimetry or ultraviolet and visible absorption spectroscopy detection. The method is convenient in operation, is good for popularization, and can fast, sensitively and selectively detect the melamine.
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 oligonucleotides-modified nanometer gold.
Background technology
In oligonucleotides-modified nano-Au solution, when adding complementary target oligonucleotide generation hybridization, will cause nanometer gold to be reunited, follow the variation of solution colour and absorbance, thereby realize detection target oligonucleotide.Oligonucleotides-modified nanometer gold has very high optical extinction coefficient, and the color of solution and the distance between nano Au particle are closely related.By specific oligonucleotide connecting arm, can be with this principles in different reaction systems.The present invention is based upon on the basis of this principle just.Not long ago, the someone reported a kind of melamine detection method (Kelong Ai, Yanlan Liu, and Lehui Lu, Journal of the American ChemicalSociety, 2009,131 (27), 9496-9497.).In this method, the SR that is used for the marking nano gold is a kind of verivate of tricyanic acid, not commercialization as yet; Need oneself synthetic and purifying, this has just proposed very high requirement to operator, and not only labour intensity is big; Waste time and energy, and its purity directly influences the sensitivity and the selectivity of detection.The used sulfhydrylation oligonucleotide of the present invention is a commercialization reagent, and the detected result favorable reproducibility is beneficial to and promotes the use of.
Summary of the invention
The technical problem that the present invention will solve is exactly to overcome to have now to need expensive large-scale instrument in the method that detects trimeric cyanamide; Be difficult to realize on-line monitoring; Be unfavorable for the defective promoted, the objective of the invention is to be to provide a kind of and detect the method for trimeric cyanamide based on oligonucleotides-modified nanometer gold, easy fast; High specificity, highly sensitive.
The inventor is through discovering that single stranded oligonucleotide is the random coil state, can be adsorbed on the nanometer gold surface in solution; Specific oligonucleotide sequence can combine with some molecular specificity, and selectivity is good, and is highly sensitive.Therefore can oligonucleotide one end be used sulfydryl modification, be connected to the nanometer gold surface, constitute the nanometer gold oligonucleotide probe of stable detection target molecule through the S-Au covalent effect.When target molecule existed, target molecule combined with base specific, formed reticulated structure; Distance reduces between nm gold particles, causes the nano-Au solution change in color, directly through the observation of bore hole colorimetric; Or, can realize that target molecule detects by ultraviolet-visible spectrophotometer.The inventor has proposed new detection strategy: the character of utilizing the nano-Au solution color to change with the nm gold particles changes in spacing; It is developed into the probe that detects trimeric cyanamide; Can realize highly sensitive detection easily, thereby accomplish the present invention trimeric cyanamide.
Therefore, the present invention solves the problems of the technologies described above the technical scheme that is adopted and is: a kind of method based on oligonucleotides-modified nanometer gold detection trimeric cyanamide can may further comprise the steps successively:
1) can modify the nanometer gold surface with trimeric cyanamide specificity bonded oligonucleotide (sulfydryl modification contain a plurality of thymus pyrimidines, base adds up to 2～100, the thymus pyrimidine number is 2～100 oligonucleotide, the worker is given birth in Shanghai);
2) add trimeric cyanamide (melamine, sigma company) standardized solution or sample solution) react;
3) adopt visual colorimetry or ultraviolet visible spectrophotometry to realize to melamine detection.
Step 1) of the present invention is can be connected to the nanometer gold surface with trimeric cyanamide specificity bonded oligonucleotide to form capture probe.The present invention can realize the detection by quantitative of trimeric cyanamide, the detection limit requirement that can be up to state standards.The particle diameter of described nanometer gold is 10～20nm, and the reaction density of nano-Au solution is 1～100nM.Temperature of reaction is 10～38 ℃, and the reaction times is 50～60 hours.Oligonucleotide of the present invention is the single stranded oligonucleotide with specific base sequence; This single stranded oligonucleotide has special nucleic acid construct; Can combine with the trimeric cyanamide specificity, thereby form crosslinked nanometer gold network structure, nanometer gold precipitates rapidly under this structure; Cause that solution colour shoals, thereby realize melamine detection.The concentration ratio of described nanometer gold and oligonucleotide is 1: 50～1: 150.Similar with domestic method, step 1) is described to be contained in the oligonucleotides-modified nanometer gold reaction system of sulfydryl, needs to add buffered soln, and this buffered soln is PBS or Tris-acetate or arsonate, and reaction density is 10～20mM.Need add NaCl simultaneously to better meet the modification of oligonucleotide to nanometer gold, reaction density is 0.1～1.0M.Introduce 1 μ M～1mM halfcystine or gsh in the reaction process, can improve the stability of nanometer gold oligonucleotide probe.
Step 3) of the present invention is for step 2) reaction solution be cooled to room temperature, dilution (5～50 times) is through visual colorimetry or measure ultraviolet-visible absorption spectroscopy and realize melamine detection.Solution colour shoals, and contains trimeric cyanamide; Color is shallow more, and content of melamine is many more; 520nM place absorbancy reduces, and solution to be measured contains trimeric cyanamide, is positive.
The present invention adds the interfering substance that possibly exist in the milk food prod to step 2) sample solution in carry out control experiment.
Whole reaction system of the present invention can be controlled in the microlitre rank, and volume is 20～800 microlitres, and the optimization experiment scheme can be explained as follows: (concentration is 10 to get the specific oligonucleotide of 280uL sulfydryl modification
-3～10
-6M) slowly join the 60.0mL nano-Au solution (particle diameter 10～20nm, reaction density is at 1～100nM; The molar ratio scope of Au: DNA is 1: 50～150) in, hatched 10～20 hours, add another sulfydryl small molecules of 90.0mL buffered soln dissolved then; Continued to hatch 30～50 hours; Utilize buffered soln to clean, concentrate, obtain the nanometer gold oligonucleotide probe.Buffered soln is Tris or PBS or arsonate buffered soln commonly used, and NaCl concentration is 0.1～1.0M.In 10uL nanometer gold oligonucleotide probe solution, adding 20uL concentration is 10
-3～10
-7The melamine solution of M mixes, and is heated to 95 ℃ of afterreactions 2～10 minutes, slowly drops to room temperature (23～26 ℃).Not add one group of melamine solution, some groups of the interfering substance that possibly exist in the interpolation milk as contrast simultaneously.In above-mentioned solution, add the 400uL deionized water then respectively, through visual colorimetry or utilize uv absorption spectrum to detect.
Ultimate principle of the present invention can be summarized as: utilize oligonucleotide to combine with the trimeric cyanamide specificity; Nano-Au solution color and nm gold particles dispersion state are closely related; Reach the character that oligonucleotide can be connected to the nanometer gold surface through sulfydryl, realized melamine detection.At first design the Nano-Au probe that this specific oligonucleotide is modified, add trimeric cyanamide after, combine to form crosslinked nanometer gold network structure through trimeric cyanamide and base.The dispersive nano-Au solution presents redness, and reunite back purpling or change are blue, very soon deposition; Nano-Au solution after the reunion significantly reduces at the absorbance at the corresponding ultraviolet characteristic absorption wavelength place of mono-dispersed nano gold.Detection schematic diagram of the present invention can be referring to Fig. 1.
Compared with prior art, advantage of the present invention is following:
1) the present invention has changed detection melamine detection pattern commonly used at present, need not to use expensive large-scale instrument, has practiced thrift cost.Compare (KelongAi, Yanlan Liu, and Lehui Lu with the method for the detection trimeric cyanamide of reporting for the previous period; Journal of the American Chemical Society 2009,131 (27), 9496-9497.); Use commercial sulfhydrylation oligonucleotide marking nano gold, need not oneself synthetic SR, avoided the experimental result poor reproducibility of bringing because of synthetic technology is immature; Detect problems such as error is big, can realize the accurate detection of actual sample.
2) detection method of the present invention has high degree of specificity, can effectively avoid the interference of possibility coexisting substances in the actual sample, and is practical.
3) detection method of the present invention is highly sensitive, and detection limit can reach 46.5nM, and requirement is up to state standards.
4) the present invention detects fast.The nanometer gold oligonucleotide probe is stable, easy to store, is easy to realize commercialization production in batches, directly adopts this oligonucleotides-modified Nano-Au probe that trimeric cyanamide is detected, and whole testing process is no more than 10 minutes.
5) this law is measured the linearity range 4.65 * 10 of trimeric cyanamide
-7～4.65 * 10
-4, linearly dependent coefficient 0.9890.
Description of drawings
Below in conjunction with description of drawings feature and advantage of the present invention.
Fig. 1 is a detection principle schematic of the present invention.
Fig. 2 is the uv absorption spectra (A) that the present invention is used to detect trimeric cyanamide, working curve (B), visual colorimetry observations (C) and interfering ion allow multiple (D).
Embodiment
With the present invention the practical implementation that trimeric cyanamide detects is further specified the present invention below.The experimental technique of unreceipted actual conditions wherein is usually according to the normal condition or the condition of advising according to manufacturer.Room temperature among the present invention is meant the laboratory temperature that carries out implementation and operation, is 23～26 ℃.
Ultraviolet-visible spectrum detects: (SHIMADZU, Japan), scanning wavelength scope: 400～750nm uses the 600uL quartz colorimetric utensil to UV-2550 Spectrophotometer, gets the 430uL reaction solution and measures.
Used nanometer gold reference (K.C.Grabar, R.G.Freeman, M.B.Hommer, M.J.Natan, Anal Chem 1995,67,735-743) preparation during the present invention implements.Concrete steps are following, the 100mL ultrapure water is heated to the boiling back adds 1.88mL 1% (w/w) chlorauric acid solution.The boiling back adds 10.0mL trisodium citrate aqueous solution (quality of trisodium citrate is 0.1141g) rapidly and continues heated and stirred 10min under vigorous stirring once more, and solution becomes wine red.Stop to heat the back and stir 15min, leave standstill cool to room temperature.Use 0.22 μ m membrane filtration then, be placed on 4 ℃ of preservations in the refrigerator.It is 10～20nm that present method obtains particle diameter, and concentration is the nano-Au solution of 3～10nM.
Used oligonucleotide sequence is the single stranded oligonucleotide of 10 thymus pyrimidines among the present invention.The one of which end is modified by sulfydryl, is used to be connected to the nanometer gold surface.
Step: get 280uL 10
-4The specific oligonucleotide of M sulfydryl modification slowly joins 60.0mL nano-Au solution (particle diameter 13nm, concentration 4.67nM; The mol ratio of Au and DNA is 1: 100) in, hatched 16 hours, (10mM PBS, pH 7.4,0.1M NaCl, 10 to add 90.0mL buffered soln then
-4The ML-halfcystine), continue to hatch 40 hours.The centrifugal supernatant that goes, (10mM PBS 0.1MNaCl) cleans twice, concentrates, and obtains the nanometer gold oligonucleotide probe to utilize buffered soln.In 10uL nanometer gold oligonucleotide probe solution, adding 20uL concentration is 10
-3～10
-7The PBS of M (containing 0.3M NaCl) dissolved melamine solution mixes, be heated to 95 ℃ the reaction 5min after, slowly drop to room temperature.Not add one group of melamine solution, some groups of the interfering ion that possibly exist in the adding milk as contrast simultaneously.In above-mentioned solution, add the 400uL deionized water then respectively, detect through visual colorimetry observation or uv absorption spectrum.
Result: according to principle shown in Figure 1 and operation steps; Uv absorption spectra is seen Fig. 2 A; Fig. 2 B is a working curve; The absorbance difference that is employed in 520nm place and 700nm place characterizes, and Fig. 2 C is corresponding visual colorimetry observations, and Fig. 2 D is to can compatible interfering ion experimental result in the milk sample.It is thus clear that trimeric cyanamide can combine with nanometer gold oligonucleotide probe specificity, causes the change of nanometer gold dispersion state.No matter change with melamine concentration, be that direct visual colorimetry is observed, and still can both realize melamine detection in the sample by ultraviolet-visible pectrophotometer, detects and be limited to 46.5nM, can reach the GB requirement, linearity range 4.65 * 10
-7～4.65 * 10
-4, linearly dependent coefficient 0.9890.The present invention is practical, can effectively avoid in the actual sample maybe coexisting substances to the interference that trimeric cyanamide detects, allow that the coexistence multiple reaches several thousand times even up to ten thousand times.
Claims (1)
1. the method based on oligonucleotides-modified nanometer gold detection trimeric cyanamide the steps include:
1) can be connected to the nanometer gold surface with trimeric cyanamide specificity bonded oligonucleotide and form capture probe, the particle diameter of described nanometer gold is 10～20nm, and the reaction density of nano-Au solution is 1～100nM; Temperature of reaction is 10～38 ℃; Reaction times is 50～60 hours, and the concentration ratio of described nanometer gold and oligonucleotide is 1: 50～1: 150, in the described oligonucleotides-modified nanometer gold reaction system; Add buffered soln; This buffered soln is PBS or Tris-acetate, and reaction density is 10～20mM, adds NaCl simultaneously to satisfy the modification of oligonucleotide to nanometer gold better; Reaction density is 0.1～1.0M, introduces 1 μ M～1mM halfcystine or gsh in the reaction process;
2) react in the oligonucleotides-modified Nano-Au probe solution of step 1), adding solution to be measured, wherein, the reaction density of described solution to be measured is 10
-3～10
-7M, temperature of reaction is 70～100 ℃, and the reaction times is 3～7 minutes, and used buffered soln is Tris or PBS, or arsonate buffered soln, and reaction density is 1～100mM, adds NaCl simultaneously to satisfy combining of trimeric cyanamide and oligonucleotide better;
3) for step 2) reaction solution cool off dilution, through visual colorimetry or measure ultraviolet-visible absorption spectroscopy and realize to melamine detection;
Described oligonucleotide is that an end is by the single stranded oligonucleotide of 10 thymus pyrimidines of sulfydryl modification.
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| 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 |
| CN102661950A (en) * | 2012-05-31 | 2012-09-12 | 福建省产品质量检验研究院 | Test card for quickly detecting melamine |
| CN107300551A (en) * | 2017-06-13 | 2017-10-27 | 中国科学院上海应用物理研究所 | A kind of melamine visible detection method based on nano Au particle color change |
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Non-Patent Citations (1)
| Title |
|---|
| Kelong Ai et al.Hydrogen-Bonding Recognition-Induced Color Change of Gold Nanoparticles for Visual Detection of Melamine in Raw Milk and Infant Formula.《JACS》.2009,第131卷第9496－9497页. * |
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