CN106501243A - A kind of method of use molecular engram test strips quick detection tripolycyanamide - Google Patents
A kind of method of use molecular engram test strips quick detection tripolycyanamide Download PDFInfo
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- 238000012360 testing method Methods 0.000 title claims abstract description 67
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 21
- 229920000877 Melamine resin Polymers 0.000 claims abstract description 46
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 claims abstract description 44
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 24
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 24
- 235000013336 milk Nutrition 0.000 claims abstract description 20
- 239000008267 milk Substances 0.000 claims abstract description 20
- 210000004080 milk Anatomy 0.000 claims abstract description 20
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 19
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 19
- 238000002372 labelling Methods 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 229920000642 polymer Polymers 0.000 claims abstract description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- GVKCHTBDSMQENH-UHFFFAOYSA-L phloxine B Chemical compound [Na+].[Na+].[O-]C(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 GVKCHTBDSMQENH-UHFFFAOYSA-L 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 238000004458 analytical method Methods 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 11
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 230000012447 hatching Effects 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000000975 dye Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 239000000020 Nitrocellulose Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 229920001220 nitrocellulos Polymers 0.000 claims description 6
- 230000003647 oxidation Effects 0.000 claims description 6
- 238000007254 oxidation reaction Methods 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 239000007974 sodium acetate buffer Substances 0.000 claims description 6
- 239000002156 adsorbate Substances 0.000 claims description 5
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical class C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims description 5
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- 239000003643 water by type Substances 0.000 claims description 5
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 4
- 241000872931 Myoporum sandwicense Species 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 150000001412 amines Chemical class 0.000 claims description 2
- 238000004737 colorimetric analysis Methods 0.000 claims description 2
- 230000001419 dependent effect Effects 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 235000009392 Vitis Nutrition 0.000 claims 1
- 241000219095 Vitis Species 0.000 claims 1
- 239000007864 aqueous solution Substances 0.000 claims 1
- 229940088598 enzyme Drugs 0.000 claims 1
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 claims 1
- 238000007689 inspection Methods 0.000 claims 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 6
- 238000006116 polymerization reaction Methods 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 5
- 239000012528 membrane Substances 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000007421 fluorometric assay Methods 0.000 abstract 1
- 230000035484 reaction time Effects 0.000 abstract 1
- 235000001727 glucose Nutrition 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 7
- 239000012086 standard solution Substances 0.000 description 5
- 235000013350 formula milk Nutrition 0.000 description 4
- 150000002304 glucoses Chemical class 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001506 fluorescence spectroscopy Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
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- 230000002860 competitive effect Effects 0.000 description 2
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- 230000036541 health Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- -1 triazines nitrogen heterocyclic ring organic compound Chemical class 0.000 description 2
- 238000005829 trimerization reaction Methods 0.000 description 2
- 208000000913 Kidney Calculi Diseases 0.000 description 1
- 239000004640 Melamine resin Substances 0.000 description 1
- 206010029148 Nephrolithiasis Diseases 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 208000006568 Urinary Bladder Calculi Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 235000014510 cooky Nutrition 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010891 electric arc Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229920000344 molecularly imprinted polymer Polymers 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a kind of method of use molecular engram test strips quick detection tripolycyanamide.The melamine molecular engram polymer that chemical polymerization is prepared is fixed in test strips, the tripolycyanamide of tripolycyanamide and glucoseoxidase labelling in sample is carrying out compound reaction time generation competition with trace hole, the tripolycyanamide of the glucoseoxidase labelling on reaction molecular blotting membrane is replaced by tripolycyanamide, with the minimizing of glucoseoxidase, the oxidized amount for producing hydrogen peroxide of glucose in the liquid of bottom is reduced, the oxidized amount of phloxine is also reduced, during using Fluorometric assay, fluorescence intensity also reduces accordingly, in sample, the concentration of tripolycyanamide is 1.0 × 10‑9mol/L~1.0×10‑4With the decrement of fluorescence intensity into good logarithmic relationship in the range of mol/L, detection is limited to 9.0 × 10‑10Mol/L, it is achieved that the quick detection to tripolycyanamide.The present invention has simple, quick, with low cost, accurately and reliably, it is adaptable to the quick detection of the tripolycyanamide in milk sample.
Description
Technical field
The present invention relates to molecular imprinting field, and in particular to a kind of with molecular engram test strips quick detection melamine
The method of amine.
Background technology
Tripolycyanamide（MEL）It is a kind of triazines nitrogen heterocyclic ring organic compound, abbreviation triamine, molecular weight are 126.13.
It is a kind of colourless monoclinic prism body, tasteless, the dissolubility in water and in organic solvent is all very low.Tripolycyanamide is a kind of purposes
Extensive Organic chemical products, used as production melamine resin（MF）Raw material be its topmost purposes, containing trimerization
The Chemical products of cyanamide have nonflammable, water-fast, heat-resisting, resistance to electric arc, the advantage of ageing-resistant, resistance to chemical attack.
Tripolycyanamide and its derivant are not protein in itself, can not only provide nutrition to health on the contrary to body
Body health is adversely affected.But because its nitrogen content is higher, 66% is reached, often illegally added the conventional foods such as milk, cookiess
In, such as in September, 2008, illegal interpolation tripolycyanamide contamination accident in the baby milk powder of China's outburst.
After adding tripolycyanamide in milk, illusion that protein content can only be caused in the detection to increase long-term repeats
A large amount of intake tripolycyanamide may cause vesical calculuses, renal calculuss etc..Food and Drug Administration office（FDA）Set tripolycyanamide
Everyone tolerates intake daily（TDI）For 0.63 mg/kg body weight.The safe threshold value of tripolycyanamide in baby milk powder is required
More strict, daily tolerance intake is set as 1/10th of adult, body weight is calculated with 7.0 kg, then infant formula milk
Safety value in powder is 0.0441 mg.Therefore, in milk, the detection of tripolycyanamide has great importance.And tradition evaluates cattle
The Kjeldahl's method of milk quality can only determine the total amount of nitrogen, but can not effectively distinguish source and the species of nitrogen.Conventional trace
Tripolycyanamide analysis method mainly includes high performance liquid chromatography (HPLC), gas chromatography-mass spectrography detection method (GC-MS), height
Effect liquid phase chromatogram-mass spectrography（HPLC-MS）, enzyme linked immunosorbent assay（ELISA）, more or less all there is instrument in these methods
The shortcomings of operation is complex, loaded down with trivial details, the time is longer, it is difficult to meet the requirement of field or the detection of mass field rapid screening.
In tripolycyanamide detection method, in the urgent need to a kind of method for quick with more high sensitivity and detection limit, and molecule
The characteristics of high selectivity that engram technology has and high sensitivity, if combined with quick detection test paper so as to setting up to melamine
Amine is more simple and efficient and sensitivity to meet the method for routine analysis requirement be that there is very good prospect and practical application to anticipate
Justice.
Content of the invention
The purpose of the present invention is there is complex pretreatment, time length, cost for the detection method of existing tripolycyanamide
A kind of high present situation, there is provided method for quick of tripolycyanamide, the method is simple, quick, low cost.The present invention can be used for cattle
The quick analysis detection of tripolycyanamide in milk sample.
Concretely comprise the following steps：
(1) preparation of melamine molecular engram polymer
0.0020 ~ 0.0032 g N,N methylene bis acrylamides are weighed, it is 3 × 10 to add 20 mL concentration-4Mol/L's
Tripolycyanamide（MEL）Solution and 2 ~ 7mL analyze pure α-methacrylic acid, and ultrasound 10 minutes, after standing overnight, adds 0.01
~ 0.03g Ammonium persulfate .s cause, and react 2 ~ 7 minutes, that is, melamine molecular engram polymer is obtained（MIP）, in refrigerator
Storage, standby.
(2) periodate oxidation method prepares glucoseoxidase labelling tripolycyanamide (GOD-MEL)
Weigh 4 ~ 6 mg glucoseoxidases（GOD）1 mL secondary waters are dissolved in, it is 0.1 mol/L to add 0.2 mL concentration
NaIO4Solution, under room temperature, lucifuge is stirred 20 minutes；Above-mentioned solution is loaded bag filter（MW4000）In, to 1 mmol/L pH=
4.4 HAC-NaAC buffer dialysis, 4 DEG C overnight, and 20 mL concentration of addition are 0.2 mol/L pH=9.5 carbonate buffer
Liquid, makes the pH of GOD be increased to 9.0 ~ 9.5；Another 10 mL color comparison tube is taken, adds 8 ~ 12 mg tripolycyanamide and 1 mL dense
The carbonate buffer solution for 0.01 mol/L, pH=9.5 is spent, is mixed with above-mentioned GOD, room temperature lucifuge is stirred 2 hours；Toward mixing
The NaBH that 0.1 mL concentration is 4 mg/mL is added in liquid4Mix, 4 DEG C are placed 2 hours；Above-mentioned solution is loaded in bag filter,
It is the PBS dialysis of 0.15 mol/L, pH=7.4 to concentration, 4 DEG C overnight；After the completion of dialysis, by label subpackage
Enter in 1 mL color comparison tubes, 4 DEG C keep in dark place.
(3) making of test strips
4 × 1 cm of clip（Long × wide）Plastic-substrates, in the hole area that black basal region carves 0.5 × 0.5 cm
Domain, by nitrocellulose filter（NC films）The hole region back side is attached to, by step（1）Obtained melamine molecular engram polymerization
Thing Deca is dried under the conditions of room temperature after being evenly distributed on NC films surface, then, 15 ~ 25 mL mass percentages of Deca toward on MIP
Specific concentration is 25% ethanol, and eluting 2 ~ 4 minutes uses secondary water wash, to remove in melamine molecular engram polymer
Tripolycyanamide and partial adsorbates thing, dry, so as to prepare the melamine molecular engram for remaining with specific recognition hole
Test strips, keep in dark place.
(4) detection method
Take step（3）Obtained melamine molecular engram test strips one, by GOD-MEL solution good for 15 ~ 25 μ L labellings
It is added drop-wise in the molecular engram test strips for having removed template molecule, incubation 5 minutes is used secondary water wash, washes off and do not incubate
Change GOD-MEL up and other impurity, competition process is carried out in the sample of variable concentrations MEL, 15 ~ 25 μ of Deca
In test strips of the L testing sample solutions to after hatching, react 3 minutes, rinsed with secondary water；Test strips Deca 15 after by competition
The dyestuff bottom liquid of ~ 25 μ L（It is 3 × 10 comprising concentration-4The phloxine of mol/L, concentration are the glucose of 0.02 g/mL, dense
Spend the HAc-NaAc buffer for 0.1 mol/L）, carry out chromogenic reaction.The maximum excitation wavelength for adjusting fluorophotometer is 515
Nm, exciting light (EX) slit width be 1.5 nm, launching light (EM) slit width be 3.0 nm, by chromogenic reaction after test strips
Being placed in fluorophotometer carries out fluorescence intensity detection, realizes quantitative analyses.In addition, for the on-line monitoring of batch samples can
Complete by semi-quantitative analyses, i.e., with the naked eye color is simply judged（Optical colorimetry）Or taken pictures with uviol lamp
Contrast.
(5) drafting of working curve
Take step（3）The melamine molecular engram test strips for preparing, by step（4）Tripolycyanamide standard to variable concentrations
Solution carries out fluoroscopic examination, records fluorescence intensity, drawing curve.By the logarithmic regressions analysis to working curve,
The fluorescence intensity difference and 1 × 10 of test strips-8mol/L ~ 1×10-4The logarithm of mol/L melamine concentrations is linear,
Equation of linear regression isΔI F = 61.19lgC+ 572.83, linearly dependent coefficient reaches 0.9944.
(6) in milk sample content of melamine measure
The commercially available milk samples of 300 ~ 600 μ L are pipetted, is settled to the acetic acid sodium acetate buffer of 0.1 mol/L pH=5
10 mL, used as testing sample.Take step（3）Obtained melamine molecular engram test strips one, will be good for 20 μ L labellings
GOD-MEL solution is added drop-wise in the molecular engram test strips for having removed template molecule, incubation 5 minutes, is drenched with secondary water
Wash, wash the GOD-MEL and other impurity that does not hatch up off.Test strips of the 20 μ L testing sample solutions of Deca to after hatching
On, react 3 minutes, rinsed with secondary water.The dyestuff bottom liquid of the 20 μ L of test strips Deca after by competition（It is 3 comprising concentration
×10-4The HAc-NaAc bufferings that the phloxine of mol/L, concentration are the glucose of 0.02 g/mL, concentration is 0.1 mol/L
Liquid）, carry out chromogenic reaction.Test strips after by chromogenic reaction are placed in fluorophotometer and carry out fluorescence intensity detection, according to work
The concentration of tripolycyanamide in composition line computation sample.
The present invention obtains the molecularly imprinted polymer of tripolycyanamide using chemical method polymerization, is prepared into and tripolycyanamide is had
The molecular engram test strips of specific recognition capability, the hole on test strips reaction zone surface can recognize tripolycyanamide and tool simultaneously
There is the tripolycyanamide of mutually isostructural glucoseoxidase labelling.By the test strips of eluted template molecule high concentration glucose
After hatching in the tripolycyanamide of oxidase label, trace hole is occupied by the tripolycyanamide of glucoseoxidase labelling.Subsequently competing
Strive, trimerization
Cyanamide is capable of the tripolycyanamide of competitive substitution glucoseoxidase labelling and occupies again hole.With tripolycyanamide point in sample
The increase of sub- concentration, more by the tripolycyanamide of the glucoseoxidase labelling of competitive substitution on blotting membrane, that is, remain in print
Glucoseoxidase on mark film is fewer.And the glucoseoxidase on blotting membrane surface can be occurred with the glucose in the liquid of bottom
Enzyme digestion reaction produces hydrogen peroxide, and the phloxine in the further oxidation Decomposition bottom liquid of hydrogen peroxide makes which fade, therefore, passes through
The fluorescence intensity of spectrofluorophotometer test strip, realizes the fast qualitative of tripolycyanamide in sample, quantitatively or semi-quantitatively
Analysis detection.
The invention has the beneficial effects as follows：
1. the present invention detects tripolycyanamide using molecular engram test strips and fluorescence spectrophotometry, overcomes existing three
The present situations such as poly cyanamid detection method complex pretreatment, time length, cost height, the method have simple, quick, low cost, accurately
Reliable the advantages of, can be used for the online trace detection of tripolycyanamide in milk product, realize the food safety production to milk product
The purpose of supervision.
2. measuring samples solution of the present invention avoids the pretreatment process of complex sample.
3. detection method sensitivity is high, 1.0 × 10-9mol/L ~ 1.0×10-4Mol/L concentration ranges
Interior, in good linear relationship, the efficient recovery rate of detection method is the negative logarithm of fluorescence intensity difference and concentration of standard solution
91.2 % ~ 112.05 %, minimum detectability are 9.0 × 10-10mol/L（3δb/ K, δbMark for 11 blank sample detections
Quasi- deviation；Slopes of the K for working curve）.
Description of the drawings
Fig. 1 is working curve standard fluorescence colorimetric card of the present invention.
In figure：Working curve ultraviolet picture be followed successively by from left to right blank group, 1.0 × 10-9mol/L、1.0×10-8mol/
L、1.0×10-7mol/L、1.0×10-6mol/L、1.0×10-5mol/L、1.0×10-4mol/L.
Fig. 2 is the graph of a relation of tripolycyanamide standard solution content of the present invention and fluorescence developing intensity.
In figure：Melamine concentration a ~ g is respectively：1.0×10-4mol/L、1.0×10-5mol/L、1.0×10- 6mol/L、1.0×10-7mol/L、1.0×10-8mol/L、1.0×10-9Mol/L, blank group.
Fig. 3 is the calibration graph of melamine concentration of the present invention.
Specific embodiment
Below in conjunction with the accompanying drawings and specific embodiment the invention will be further described, but do not limit the present invention use model
Enclose and range of application.
Embodiment 1：
（1）The preparation of melamine molecular engram polymer
0.0020 g N,N methylene bis acrylamides are weighed, it is 3 × 10 to add 20 mL concentration-4The tripolycyanamide of mol/L
（MEL）Solution and 2mL α-methacrylic acids, ultrasound 10 minutes, after standing overnight, add 0.013g Ammonium persulfate .s to cause, reaction
2 minutes, that is, melamine molecular engram polymer is obtained（MIP）, store in refrigerator, standby.
（2）Periodate oxidation method prepares glucoseoxidase labelling tripolycyanamide (GOD-MEL)
Weigh 4 mg glucoseoxidases（GOD）1 mL secondary waters are dissolved in, the NaIO that 0.2 mL concentration is 0.1 mol/L is added4
Solution, under room temperature, lucifuge is stirred 20 minutes；Above-mentioned solution is loaded bag filter（MW4000）In, to 1 mmol/L pH=4.4
HAC-NaAC buffer dialysis, 4 DEG C are overnight, add 20 mL, 0.2 mol/L pH=9.5 carbonate buffer solutions, make GOD
PH be increased to 9.0；Another 10 mL color comparison tube is taken, 0.01 mol/L pH=9.5 of 8 mg tripolycyanamide and 1 mL are added
Carbonate buffer solution, mix with above-mentioned GOD, room temperature lucifuge stir 2 hours；It is 4 to add 0.1 mL concentration toward mixed liquor
The NaBH of mg/mL4Mix, 4 DEG C are placed 2 hours；Above-mentioned solution is loaded in bag filter, to 0.15 mol/L pH=7.4
PBS is dialysed, and 4 DEG C overnight；After the completion of dialysis, label is distributed in 1 mL color comparison tubes, 4 DEG C keep in dark place.
（3）The making of test strips
4 × 1 cm of clip（Long × wide）Plastic-substrates, in the hole area that black basal region carves 0.5 × 0.5 cm
Domain, by nitrocellulose filter（NC films）The hole region back side is attached to, by step（1）Obtained melamine molecular engram polymerization
Thing Deca is dried under the conditions of room temperature after being evenly distributed on NC films surface, then, the 15 mL mass percent concentrations of Deca toward on MIP
For 25% ethanol, eluting 2 minutes, use secondary water wash, with remove the tripolycyanamide in melamine molecular engram polymer and
Partial adsorbates thing, dries, so as to prepare the melamine molecular engram test strips for remaining with specific recognition hole, lucifuge
Preserve.
（4）The preparation of testing sample solution：Precision pipettes 1.0 mL milk samples to be checked in 10 mL color-comparison tubes,
With the NaAc_HAc buffer solution constant volume of 0.1 mol/L pH=5, as sample sets；Parallel 1.0 mL that pipette are with batch cattle
Milk sample adds 0.5 mL 1.0 × 10 into 10 mL color-comparison tubes-4Mol/L tripolycyanamide standard solution, uses 0.1
The sodium acetate buffer constant volume of mol/L pH=5 as mark-on group, every group of parallel assay three times.
（5）Take step（3）Obtained melamine molecular engram test strips, 20 μ L GOD- of Deca in its reaction zone
MEL, incubation 5 minutes, with 10 mL, bis- water wash, wash the GOD-MEL and other impurity that does not hatch up off.
（6）By 20 μ L steps（4）The testing sample of sample sets is added drop-wise to step（5）In test strips after hatching, reaction 3
Minute, rinsed with secondary water.
（7）The dyestuff bottom liquid of the 20 μ L of test strips Deca after by competition（It is 3 × 10 comprising concentration-4Mol/L fluorescence Fructus Persicae
Red, concentration is 0.02g/mL glucoses, the HAc-NaAc buffer that concentration is 0.1 mol/L）, carry out chromogenic reaction 3 minutes.
（8）Take fluorescence cuvette, by chromogenic reaction after test strips be placed in RF5301 fluorophotometers that to carry out fluorescence strong
Degree detection, the system adopt maximum excitation wavelength to be respectively 1.5 nm, 3.0 nm for the slit width of 515 nm, EX and EM, enter
Row fluorescence intensity is detected, and with the naked eye color can be carried out simply judging and being taken pictures with uviol lamp during semi-quantitative analyses,
Compare with fluorescence standard colorimetric card.
（9）Ibid, collection fluorescence data carries out recovery of standard addition experiment to mark-on sample sets assay method.
Embodiment 2：
（1）The preparation of melamine molecular engram polymer
0.003 g N,N methylene bis acrylamides are weighed, it is 3 × 10 to add 20 mL concentration-4The tripolycyanamide of mol/L
（MEL）Solution and 5mL α-methacrylic acids, ultrasound 10 minutes, after standing overnight, add 0.02g Ammonium persulfate .s to cause, reaction 5
Minute, that is, melamine molecular engram polymer is obtained（MIP）, store in refrigerator, standby.
（2）Periodate oxidation method prepares glucoseoxidase labelling tripolycyanamide (GOD-MEL)
Weigh 5 mg glucoseoxidases（GOD）1 mL secondary waters are dissolved in, the NaIO that 0.2 mL concentration is 0.1 mol/L is added4
Solution, under room temperature, lucifuge is stirred 20 minutes；Above-mentioned solution is loaded bag filter（MW4000）In, to 1 mmol/L pH=4.4
HAC-NaAC buffer dialysis, 4 DEG C are overnight, add 20 mL, 0.2 mol/L pH=9.5 carbonate buffer solutions, make GOD
PH be increased to 9.5；Another 10 mL color comparison tube is taken, 0.01 mol/L pH=9.5 of 10 mg tripolycyanamide and 1 mL are added
Carbonate buffer solution, mix with above-mentioned GOD, room temperature lucifuge stir 2 hours；It is 4 to add 0.1 mL concentration toward mixed liquor
The NaBH of mg/mL4Mix, 4 DEG C are placed 2 hours；Above-mentioned solution is loaded in bag filter, to 0.15 mol/L pH=7.4
PBS is dialysed, and 4 DEG C overnight；After the completion of dialysis, label is distributed in 1 mL color comparison tubes, 4 DEG C keep in dark place.
（3）The making of test strips
4 × 1 cm of clip（Long × wide）Plastic-substrates, in the hole area that black basal region carves 0.5 × 0.5 cm
Domain, by nitrocellulose filter（NC films）The hole region back side is attached to, by step（1）Obtained melamine molecular engram polymerization
Thing Deca is dried under the conditions of room temperature after being evenly distributed on NC films surface, then, the 20 mL mass percent concentrations of Deca toward on MIP
For 25% ethanol, eluting 3 minutes, use secondary water wash, with remove the tripolycyanamide in melamine molecular engram polymer and
Partial adsorbates thing, dries, so as to prepare the melamine molecular engram test strips for remaining with specific recognition hole, lucifuge
Preserve.
（4）1.0 mLs to be checked milk sample is accurately pipetted in 10 mL color-comparison tubes, with the vinegar of 0.1 mol/L pH=5
Acid-sodium acetate buffer constant volume, used as sample sets.Parallel 1.0 mL that pipette fill in colorimetric with batch milk sample to 10 mL tools
Guan Zhong, adds 1.0 mL 1.0 × 10-6Mol/L tripolycyanamide standard solution, the sodium acetate buffer with 0.1 mol/L pH=5 are molten
Liquid constant volume as mark-on group, every group of parallel assay three times.
（5）Take step（3）The melamine molecular engram test strips of preparation, 20 μ L GOD- of Deca in its reaction zone
MEL, incubation 5 minutes, with 10 mL, bis- water wash, wash the GOD-MEL and other impurity that does not hatch up off.
（6）By 20 μ L steps（4）The testing sample of sample sets is added drop-wise to step（5）In test strips after hatching, reaction 3
Minute, rinsed with secondary water.
（7）The dyestuff bottom liquid of the 20 μ L of test strips Deca after by competition（Comprising 3 × 10-4Mol/L phloxines,
0.02 g/mL glucoses, the HAc-NaAc buffer of 0.1 mol/L）, carry out chromogenic reaction 3 minutes.
（8）Take fluorescence cuvette, by chromogenic reaction after test strips be placed in RF5301 fluorophotometers and detect its fluorescence
Intensity, slit width respectively 1.5 nm, 3.0 nm for arranging maximum excitation wavelength for 515 nm, EX and EM carry out fluorescence intensity
Detection.In addition, for semi-quantitative analyses, with the naked eye color can be carried out simply to judge and taken pictures with uviol lamp, and glimmering
Light standard colorimetric card is completed by comparing.
（9）Ibid, collection fluorescence data carries out recovery of standard addition experiment to mark-on sample sets assay method.
Embodiment 3：
（1）The preparation of melamine molecular engram polymer
0.0032 g N,N methylene bis acrylamides are weighed, it is 3 × 10 to add 20 mL concentration-4The tripolycyanamide of mol/L
（MEL）Solution and 7mL α-methacrylic acids, ultrasound 10 minutes, after standing overnight, add 0.03g Ammonium persulfate .s to cause, reaction 7
Minute, that is, melamine molecular engram polymer is obtained（MIP）, store in refrigerator, standby.
（2）Periodate oxidation method prepares glucoseoxidase labelling tripolycyanamide (GOD-MEL)
Weigh 6 mg glucoseoxidases（GOD）1 mL secondary waters are dissolved in, the NaIO that 0.2 mL concentration is 0.1 mol/L is added4
Solution, under room temperature, lucifuge is stirred 20 minutes；Above-mentioned solution is loaded bag filter（MW4000）In, to 1 mmol/L pH=4.4
HAC-NaAC buffer dialysis, 4 DEG C are overnight, add 20 mL, 0.2 mol/L pH=9.5 carbonate buffer solutions, make GOD
PH be increased to 9.5；Another 10 mL color comparison tube is taken, 0.01 mol/L pH=9.5 of 12 mg tripolycyanamide and 1 mL are added
Carbonate buffer solution, mix with above-mentioned GOD, room temperature lucifuge stir 2 hours；It is 4 to add 0.1 mL concentration toward mixed liquor
The NaBH of mg/mL4Mix, 4 DEG C are placed 2 hours；Above-mentioned solution is loaded in bag filter, to 0.15 mol/L pH=7.4
PBS dialysis, 4 DEG C are overnight；After the completion of dialysis, label is distributed in 1 mL color comparison tubes, 4 DEG C keep in dark place.
（3）The making of test strips
4 × 1 cm of clip（Long × wide）Plastic-substrates, in the hole area that black basal region carves 0.5 × 0.5 cm
Domain, by nitrocellulose filter（NC films）The hole region back side is attached to, by step（1）Obtained melamine molecular engram polymerization
Thing Deca is dried under the conditions of room temperature after being evenly distributed on NC films surface, then, the 25 mL mass percent concentrations of Deca toward on MIP
For 25% ethanol, eluting 4 minutes, use secondary water wash, with remove the tripolycyanamide in melamine molecular engram polymer and
Partial adsorbates thing, dries, so as to prepare the melamine molecular engram test strips for remaining with specific recognition hole, lucifuge
Preserve.
（4）The preparation of testing sample solution：Precision pipettes 1.0 mL milk samples to be checked in 10 mL color-comparison tubes,
With the NaAc_HAc buffer solution constant volume of 0.1mol/L pH=5, as sample sets.Parallel 1.0 mL that pipette are with batch milk
Sample adds 0.4 mL 1.0 × 10 into 10 mL color-comparison tubes-6Mol/L tripolycyanamide standard solution, uses 0.1 mol/L
The sodium acetate buffer constant volume of pH=5 as mark-on group, every group of parallel assay three times.
（5）Take step（3）Obtained melamine molecular engram test strips, 20 μ L glucoses of Deca in its reaction zone
Oxidase label melamine solution, incubation 5 minutes, with 10 mL, bis- water wash, wash the GOD- that does not hatch up off
MEL and other impurity.
（6）By 20 μ L steps（4）The testing sample of sample sets is added drop-wise to step（5）In test strips after hatching, reaction 3
Minute, rinsed with secondary water.
（7）The dyestuff bottom liquid of the 20 μ L of test strips Deca after by competition（Comprising 3 × 10-4Mol/L phloxines,
0.02g/mL glucoses, the HAc-NaAc buffer of 0.1 mol/L）, carry out chromogenic reaction 3 minutes.
（8）Take fluorescence cuvette, by chromogenic reaction after test strips be placed in RF5301 fluorophotometers that to carry out fluorescence strong
Degree detection, the system adopt maximum excitation wavelength to be respectively 1.5 nm, 3.0 nm for the slit width of 515 nm, EX and EM and carry out
Fluorescence intensity is detected, and with the naked eye color can be carried out simply judging and being taken pictures with uviol lamp during semi-quantitative analyses, with
Fluorescence standard colorimetric card is compared.
（9）Ibid, collection fluorescence data carries out recovery of standard addition experiment to mark-on sample sets assay method.
According to above-mentioned detection method and computing formula, in three batches of milk samples, interpolation tripolycyanamide, mark-on reclaims are not detected
In the range of 91.2% ~ 112.05%, relative standard deviation is 2.3% ~ 2.7% to rate.The assay method of tripolycyanamide is returned
Yield is shown in Table 1 with relative standard deviation.
Claims (1)
1. a kind of method of quick detection tripolycyanamide, it is characterised in that concretely comprise the following steps：
(1) preparation of melamine molecular engram polymer
0.0020 ~ 0.0032 g N,N methylene bis acrylamides are weighed, it is 3 × 10 to add 20 mL concentration-4Mol/L tri-
Paracyanogen amine aqueous solution, 2 ~ 7 mL analyze pure α-methacrylic acid, ultrasound 10 minutes；After standing overnight, 0.01 ~ 0.03 g is added
Ammonium persulfate. causes, and reacts 2 ~ 7 minutes, that is, melamine molecular engram polymer is obtained, stores in refrigerator, standby；
(2) periodate oxidation method prepares glucoseoxidase labelling tripolycyanamide
Weigh 4 ~ 6 mg glucoseoxidases and be dissolved in 1 mL secondary waters, add the NaIO that 0.2 mL concentration is 0.1 mol/L4Molten
Liquid, under room temperature, lucifuge is stirred 20 minutes；Above-mentioned solution is loaded in bag filter, is 1 mmol/L, pH=4.4 to concentration
HAC-NaAC buffer is dialysed, and 4 DEG C overnight；Add 20 mL concentration for 0.2 mol/L pH=9.5 carbonate buffer solution, make
The pH of glucoseoxidase is increased to 9.0 ~ 9.5；Another 10 mL color comparison tube is taken, 8 ~ 12 mg tripolycyanamide and 1 are added
ML concentration is the carbonate buffer solution of 0.01 mol/L pH=9.5, mixes with above-mentioned glucoseoxidase, and room temperature lucifuge is stirred
Mix 2 hours；The NaBH that 0.1 mL concentration is 4 mg/mL is added toward mixed liquor4Mix, 4 DEG C are placed 2 hours；Will be above-mentioned molten
Liquid loads in bag filter, is the PBS dialysis of 0.15 mol/L, pH=7.4 to concentration, and 4 DEG C overnight；Dialysis is completed
Afterwards, label is distributed in 1 mL color comparison tubes, 4 DEG C keep in dark place；
(3) making of test strips
The plastic-substrates of clip length × a width of 4 × 1 cm, in the hole area that black basal region carves 0.5 × 0.5 cm
Nitrocellulose filter is attached to the hole region back side, by step by domain（1）Obtained melamine molecular engram polymer Deca
On nitrocellulose filter surface, dry under the conditions of room temperature after being evenly distributed；Then, toward Deca 15 on tripolycyanamide imprinted polymer
~ 25 mL mass percent concentrations are 25% ethanol, and eluting 2 ~ 4 minutes uses secondary water wash, to remove tripolycyanamide point
Tripolycyanamide and partial adsorbates thing in sub- imprinted polymer, dries, remains with specific recognition hole so as to prepare
Melamine molecular engram test strips, keep in dark place；
(4) detection method
Take step（3）Obtained melamine molecular engram test strips one, will be glycoxidative for Fructus Vitis viniferae good for 15 ~ 25 μ L labellings
Enzyme labelling melamine solution is added drop-wise in the molecular engram test strips for having removed template molecule, incubation 5 minutes, uses two
Secondary water wash, washes the glucoseoxidase labelling tripolycyanamide and other impurity that does not hatch up off；Competition process is not
With carried out in the sample of concentration tripolycyanamide, in test strips of 15 ~ 25 μ L testing sample solutions of Deca to after hatching, competition
Reaction 3 minutes, is rinsed with secondary water；15 ~ 25 μ L of test strips Deca after by competition are 3 × 10 comprising concentration-4Mol/L's
The dyestuff bottom liquid of the HAc-NaAc buffer that phloxine, concentration are the glucose of 0.02 g/mL, concentration is 0.1 mol/L, enters
Row chromogenic reaction；The maximum excitation wavelength for adjusting fluorophotometer is 515 nm, exciting light is that EX slit widths are 1.5 nm, send out
Light i.e. EM slit widths are penetrated for 3.0 nm, by chromogenic reaction after test strips be placed in fluorophotometer and carry out fluorescence intensity inspection
Survey, realize quantitative analyses；In addition, can complete by semi-quantitative analyses for the on-line monitoring of batch samples, i.e., with the naked eye right
Color optical colorimetry is carried out simply judging or carries out contrast of taking pictures with uviol lamp；
(5) drafting of working curve
Take step（3）Obtained melamine molecular engram test strips, by step（4）Tripolycyanamide standard to variable concentrations is molten
Liquid carries out fluoroscopic examination, records fluorescence intensity, drawing curve；By the logarithmic regressions analysis to working curve, try
The fluorescence intensity difference and 1 × 10 of paper slip-8mol/L ~ 1×10-4The logarithm of mol/L melamine concentrations is linear, line
Property regression equation isΔI F = 61.19lgC+ 572.83, linearly dependent coefficient reaches 0.9944；
(6) in milk sample content of melamine measure
The commercially available milk samples of 300 ~ 600 μ L are pipetted, molten with the acetic acid sodium acetate buffer that concentration is 0.1 mol/L, pH=5
Liquid is settled to 10 mL, used as testing sample；Take step（3）Obtained melamine molecular engram test strips one are good by labelling
20 μ L of glucoseoxidase labelling melamine solution be added drop-wise in the molecular engram test strips for having removed template molecule,
Incubation 5 minutes, uses secondary water wash, washes the glucoseoxidase labelling tripolycyanamide do not hatched up off and others are miscellaneous
Matter；In test strips of the 20 μ L testing sample solutions of Deca to after hatching, react 3 minutes, rinsed with secondary water；After by competition
20 μ L of test strips Deca are 3 × 10 comprising concentration-4The phloxine of mol/L, concentration are the glucose of 0.02 g/mL, concentration
For the dyestuff bottom liquid of the HAc-NaAc buffer of 0.1 mol/L, chromogenic reaction is carried out；Test strips after by chromogenic reaction are placed in glimmering
Fluorescence intensity detection is carried out in light photometer, according to the concentration that working curve calculates tripolycyanamide in sample.
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