CN106501243B - A method of melamine is quickly detected with molecular engram test strips - Google Patents
A method of melamine is quickly detected with molecular engram test strips Download PDFInfo
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- CN106501243B CN106501243B CN201610869710.7A CN201610869710A CN106501243B CN 106501243 B CN106501243 B CN 106501243B CN 201610869710 A CN201610869710 A CN 201610869710A CN 106501243 B CN106501243 B CN 106501243B
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- 229920000877 Melamine resin Polymers 0.000 title claims abstract description 127
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 title claims abstract description 120
- 238000012360 testing method Methods 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 24
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 37
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 37
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 20
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 20
- 235000013336 milk Nutrition 0.000 claims abstract description 19
- 239000008267 milk Substances 0.000 claims abstract description 19
- 210000004080 milk Anatomy 0.000 claims abstract description 19
- 229920000642 polymer Polymers 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 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 8
- 239000000523 sample Substances 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000007853 buffer solution Substances 0.000 claims description 16
- 238000000502 dialysis Methods 0.000 claims description 13
- 239000000872 buffer 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
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-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
- 238000003756 stirring Methods 0.000 claims description 9
- 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 description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 230000012447 hatching Effects 0.000 claims description 8
- 239000000975 dye Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 150000007974 melamines Chemical class 0.000 claims description 6
- 229920001220 nitrocellulos Polymers 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 239000007974 sodium acetate buffer Substances 0.000 claims description 6
- 239000011837 N,N-methylenebisacrylamide Substances 0.000 claims description 5
- 239000000020 Nitrocellulose Substances 0.000 claims description 5
- 239000002156 adsorbate Substances 0.000 claims description 5
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 5
- 235000019441 ethanol Nutrition 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 239000003550 marker Substances 0.000 claims description 5
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims description 5
- 230000003647 oxidation Effects 0.000 claims description 5
- 238000007254 oxidation reaction Methods 0.000 claims description 5
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 5
- 238000012764 semi-quantitative analysis Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- 150000001412 amines Chemical class 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 2
- 240000006365 Vitis vinifera Species 0.000 claims description 2
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 2
- 238000004737 colorimetric analysis Methods 0.000 claims description 2
- 230000001419 dependent effect Effects 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims description 2
- 238000004445 quantitative analysis Methods 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
- 241000872931 Myoporum sandwicense 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
- 230000000149 penetrating effect Effects 0.000 claims 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 239000012528 membrane Substances 0.000 abstract description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000007421 fluorometric assay Methods 0.000 abstract 1
- 238000006116 polymerization reaction Methods 0.000 abstract 1
- 230000035484 reaction time Effects 0.000 abstract 1
- 235000001727 glucose Nutrition 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 6
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 4
- 235000013350 formula milk Nutrition 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000005829 trimerization reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 238000001506 fluorescence spectroscopy Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 244000144730 Amygdalus persica Species 0.000 description 1
- 208000000913 Kidney Calculi Diseases 0.000 description 1
- 239000004640 Melamine resin Substances 0.000 description 1
- 206010029148 Nephrolithiasis Diseases 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 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
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000015895 biscuits Nutrition 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000003486 chemical etching Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 235000013365 dairy product Nutrition 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
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000002304 glucoses Chemical class 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
- 230000000977 initiatory effect Effects 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
- 230000007246 mechanism Effects 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
- 238000002203 pretreatment Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- -1 triazines nitrogen heterocyclic ring organic compound Chemical class 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
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
-
- 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"
-
- 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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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods for quickly detecting melamine with molecular engram test strips.The melamine molecular engram polymer that chemical polymerization is prepared is fixed in test strips, the melamine of melamine and glucose oxidase label in sample is carrying out compound reaction time generation competition with trace hole, the melamine of glucose oxidase label on reaction molecular blotting membrane is replaced by melamine, with the reduction of glucose oxidase, glucose in the liquid of bottom is oxidized the amount reduction for generating hydrogen peroxide, the amount that phloxine is oxidized also is reduced, when using Fluorometric assay, fluorescence intensity also reduces accordingly, the concentration of melamine is 1.0 × 10 in sample‑9mol/L~1.0×10‑4With the reduction amount of fluorescence intensity at good logarithmic relationship within the scope of mol/L, detection is limited to 9.0 × 10‑10Mol/L realizes the quick detection to melamine.The present invention has many advantages, such as the quick detection of simple, quick, low in cost, accurate and reliable melamine suitable for milk sample.
Description
Technical field
The present invention relates to molecular imprinting fields, and in particular to a kind of quickly to detect melamine with molecular engram test strips
The method of amine.
Background technique
Melamine (MEL) is a kind of triazines nitrogen heterocyclic ring organic compound, abbreviation triamine, molecular weight 126.13.
It is a kind of colourless monoclinic prism body, tasteless, solubility in water and organic solvent is all very low.Melamine is a kind of purposes
Extensive Organic chemical products, the raw material as production melamine resin (MF) is its most important purposes, contains trimerization
The Chemical products of cyanamide have nonflammable, water-fast, heat-resisting, resistance to electric arc, advantage ageing-resistant, resistant to chemical etching.
Melamine and its derivative itself are not protein, cannot not only provide human health nutrition instead to body
Body health adversely affects.But because its nitrogen content is higher, reaches 66%, the conventional foods such as milk, biscuit are illegally often added
In, such as in September, 2008, China outburst baby milk powder in illegally add melamine contamination accident.
After adding melamine in milk, the illusion that protein content increases can only be caused in the detection, it is long-term to repeat
A large amount of intake melamines may cause vesical calculus, kidney stone etc..Food and Drug Enforcement Administration (FDA) set melamine
It is 0.63 mg/kg weight that everyone is resistant to intake (TDI) daily.Safe threshold value requirement to melamine in baby milk powder
It is more stringent, daily tolerance intake is set as 1/10th of adult, and weight is calculated with 7.0 kg, then infant formula milk
Safety value in powder is 0.0441 mg.Therefore, the detection of melamine has great importance in milk.And tradition evaluation ox
The Kjeldahl's method of milk quality can only measure the total amount of nitrogen, but can not effectively distinguish source and the type of nitrogen.Conventional trace
Melamine analysis method mainly includes high performance liquid chromatography (HPLC), gas chromatography-mass spectrography detection method (GC-MS), height
More or less all there is instrument in effect liquid phase chromatogram-mass spectrography (HPLC-MS), enzyme linked immunosorbent assay (ELISA), these methods
The disadvantages of complicated operation, cumbersome, the time is longer, it is difficult to meet the requirement in field or the quick selective mechanisms of mass field.
In melamine detection method, there is an urgent need to a kind of with more highly sensitive and detection limit rapid detection method, and molecule
The highly selective and highly sensitive feature that engram technology has, as can to establish to melamine in conjunction with quick detection test paper
Amine is more simple and efficient and sensitivity to meet the method for routine analysis requirement anticipated with very good prospect and practical application
Justice.
Summary of the invention
The purpose of the present invention is there are complex pretreatments, time length, cost for the detection method for existing melamine
High status, provides a kind of rapid detection method of melamine, and this method is simple, quick, at low cost.The present invention can be used for ox
The quick analysis detection of melamine in milk sample.
Specific steps are as follows:
(1) preparation of melamine molecular engram polymer
0.0020 ~ 0.0032 g N, N- methylene-bisacrylamide is weighed, it is 3 × 10 that 20 mL concentration, which are added,-4
Melamine (MEL) solution of mol/L and 2 ~ 7mL analyze pure α-methacrylic acid, and ultrasound 10 minutes after standing overnight, adds
Enter the initiation of 0.01 ~ 0.03g ammonium persulfate, reacts 2 ~ 7 minutes, obtain melamine molecular engram polymer (MIP),
It is stored in refrigerator, it is spare.
(2) periodate oxidation method prepares glucose oxidase label melamine (GOD-MEL)
It weighs 4 ~ 6 mg glucose oxidases (GOD) and is dissolved in 1 mL secondary water, 0.2 mL concentration of addition is 0.1 mol/L
NaIO4Solution is protected from light stirring 20 minutes at room temperature；Above-mentioned solution is fitted into bag filter (MW4000), to 1 mmol/L pH
=4.4 HAC-NaAC buffer dialysis, 4 DEG C overnight, and it is slow for 0.2 carbonate of mol/L pH=9.5 that 20 mL concentration are added
Fliud flushing makes the pH of GOD be increased to 9.0 ~ 9.5；Another 10 mL colorimetric cylinder is taken, 8 ~ 12 mg melamines and 1 mL is added
Concentration is the carbonate buffer solution of 0.01 mol/L, pH=9.5, is mixed with above-mentioned GOD, and room temperature is protected from light stirring 2 hours；It is past mixed
Close the NaBH for being added that 0.1 mL concentration is 4 mg/mL in liquid4It mixes, 4 DEG C are placed 2 hours；Above-mentioned solution is packed into bag filter
In, it dialyses to the PBS buffer solution that concentration is 0.15 mol/L, pH=7.4,4 DEG C are overnight；After the completion of dialysis, by marker point
It is fitted into 1 mL colorimetric cylinder, 4 DEG C are kept in dark place.
(3) production of test strips
4 × 1 cm(long of clip × wide) plastic-substrates, carve the hole of 0.5 × 0.5 cm in black basal region
Nitrocellulose filter (NC film) is attached to the hole region back side by region, and melamine molecular engram made from step (1) is poly-
Close object be added dropwise in NC film surface, dried under room temperature after being evenly distributed, then, toward MIP on dropwise addition 15 ~ 25 mL mass hundred
Dividing specific concentration is 25% ethyl alcohol, elutes 2 ~ 4 minutes, is eluted with secondary water, to remove in melamine molecular engram polymer
Melamine and partial adsorbates object, dry, thus be prepared remain with specific recognition hole melamine molecule print
Mark test strips, are kept in dark place.
(4) detection method
Melamine molecular engram test strips one is taken made from step (3), the GOD-MEL that 15 ~ 25 μ L have been marked
Solution is added drop-wise in the molecular engram test strips for having removed template molecule, incubation 5 minutes, is eluted, is washed off with secondary water
The GOD-MEL and other impurity that do not hatch up, competition process is carried out in the sample of various concentration MEL, is added dropwise 15
~ 25 μ L testing sample solutions are reacted 3 minutes, are rinsed with secondary water in the test strips to after hatching；By the test strips drop after competition
The dyestuff bottom liquid for adding 15 ~ 25 μ L (is 3 × 10 comprising concentration-4 The phloxine of mol/L, the grape that concentration is 0.02 g/mL
Sugar, the HAc-NaAc buffer that concentration is 0.1 mol/L), carry out chromogenic reaction.Adjust the maximum excitation wavelength of fluophotometer
For 515 nm, exciting light (EX) slit width be 1.5 nm, transmitting light (EM) slit width is 3.0 nm, after chromogenic reaction
Test strips are placed in progress fluorescence intensity detection in fluophotometer, realize quantitative analysis.In addition, for the online of batch samples
Monitoring can be completed by semi-quantitative analysis, i.e., with the naked eye color is simply judged (optical colorimetry) or with ultraviolet lamp into
Capable comparison of taking pictures.
(5) drafting of working curve
The melamine molecular engram test strips for taking step (3) to prepare, by step (4) to the melamine of various concentration
Standard solution carries out fluorescence detection, records fluorescence intensity, draws working curve.It can by the logarithmic regressions analysis to working curve
Know, the fluorescence intensity difference and 1 × 10 of test strips-8 mol/L ~ 1×10-4The logarithm of mol/L melamine concentration linearly closes
System, equation of linear regression areΔI F = 61.19lgC + 572.83, linearly dependent coefficient reaches 0.9944.
(6) in milk sample content of melamine measurement
300 ~ 600 commercially available milk samples of μ L are pipetted, it is fixed with the acetic acid sodium acetate buffer of 0.1 pH=5 mol/L
Hold to 10 mL, as sample to be tested.Melamine molecular engram test strips one is taken made from step (3), 20 μ L are marked
Good GOD-MEL solution is added drop-wise in the molecular engram test strips for having removed template molecule, and incubation 5 minutes, use was secondary
The GOD-MEL that do not hatch up and other impurity are washed off in water elution.Examination of the 20 μ L testing sample solutions to after hatching is added dropwise
On paper slip, reacts 3 minutes, rinsed with secondary water.The dyestuff bottom liquid of 20 μ L is added dropwise (comprising dense in test strips after competition
Degree is 3 × 10-4 The phloxine of mol/L, concentration are the glucose of 0.02 g/mL, the HAc-NaAc that concentration is 0.1 mol/L
Buffer), carry out chromogenic reaction.Test strips after chromogenic reaction are placed in progress fluorescence intensity detection, root in fluophotometer
The concentration of melamine in sample is calculated according to working curve.
The present invention polymerize to obtain the molecularly imprinted polymer of melamine using chemical method, and being prepared into has melamine
The hole of the molecular engram test strips of specific recognition capability, test strips reaction zone surface can identify melamine and tool simultaneously
There is the melamine of mutually isostructural glucose oxidase label.By the test strips of eluted template molecule high concentration glucose
After hatching in the melamine of oxidase label, trace hole is occupied by the melamine that glucose oxidase marks.It is then competing
It is carried out between the melamine for the glucose oxidase label striven on the melamine and blotting membrane reacted be in the sample, trimerization
Cyanamide is capable of the melamine of competitive substitution glucose oxidase label and occupies hole again.With melamine point in sample
The increase of sub- concentration, the melamine marked on blotting membrane by the glucose oxidase of competitive substitution is more, that is, remains in print
Glucose oxidase on mark film is fewer.And the glucose oxidase of trace film surface can occur with the glucose in the liquid of bottom
Enzyme digestion reaction generates hydrogen peroxide, and the phloxine in the further oxygenolysis bottom liquid of hydrogen peroxide passes through its colour fading, therefore
The fluorescence intensity of sepectrophotofluorometer test strip realizes the fast qualitative, quantitatively or semi-quantitatively of melamine in sample
Analysis detection.
The beneficial effects of the present invention are:
1. the present invention detects melamine using molecular engram test strips and fluorescence spectrophotometry, overcome existing
Melamine detection method complex pretreatment, the time is long, the statuses such as at high cost, this method have it is simple, quick, at low cost,
The advantages that accurate and reliable, can be used for the online trace detection of melamine in milk product, realize the food safety to dairy produce
Produce the purpose of supervision.
2. the pretreatment process that measuring samples solution of the present invention avoids complex sample.
3. detection method high sensitivity, 1.0 × 10-9 mol/L ~ 1.0×10-4Mol/L concentration range
Interior, the negative logarithm of fluorescence intensity difference and concentration of standard solution is in good linear relationship, and effective rate of recovery of detection method is
The % of 91.2 % ~ 112.05, minimum detectability are 9.0 × 10-10Mol/L(3 δb/ K, δbFor the mark of 11 blank samples detection
Quasi- deviation；K is the slope of working curve).
Detailed description of the invention
Fig. 1 is working curve standard fluorescence colorimetric card of the present invention.
In figure: working curve ultraviolet picture is followed successively by blank group, 1.0 × 10 from left to right-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 relational graph of melamine standard solution content and fluorescence developing intensity of the present invention.
In figure: melamine concentration a ~ g is respectively as follows: 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
With reference to the accompanying drawing and specific embodiment the invention will be further described, but do not limit and of the invention use model
It encloses and application range.
Embodiment 1:
(1) preparation of melamine molecular engram polymer
0.0020 g N, N- methylene-bisacrylamide is weighed, it is 3 × 10 that 20 mL concentration, which are added,-4 The trimerization of mol/L
Cyanamide (MEL) solution and 2mL α-methacrylic acid, ultrasound 10 minutes after standing overnight, are added 0.013g ammonium persulfate and cause,
Reaction 2 minutes, obtains melamine molecular engram polymer (MIP), stores in refrigerator, spare.
(2) periodate oxidation method prepares glucose oxidase label melamine (GOD-MEL)
It weighs 4 mg glucose oxidases (GOD) and is dissolved in 1 mL secondary water, it is 0.1 mol/L's that 0.2 mL concentration, which is added,
NaIO4Solution is protected from light stirring 20 minutes at room temperature；Above-mentioned solution is fitted into bag filter (MW4000), to 1 mmol/L pH=
4.4 HAC-NaAC buffer dialysis, 4 DEG C overnight, and 20 mL, 0.2 carbonate buffer solution of mol/L pH=9.5 is added, makes
The pH of GOD is increased to 9.0；Take another 10 mL colorimetric cylinder, be added 8 mg melamines and 1 mL, 0.01 mol/L pH=
9.5 carbonate buffer solution is mixed with above-mentioned GOD, and room temperature is protected from light stirring 2 hours；It is 4 that 0.1 mL concentration is added into mixed liquor
The NaBH of mg/mL4It mixes, 4 DEG C are placed 2 hours；Above-mentioned solution is fitted into bag filter, to 0.15 pH=7.4 mol/L
PBS buffer solution dialysis, 4 DEG C overnight；After the completion of dialysis, marker is distributed into 1 mL colorimetric cylinder, 4 DEG C are kept in dark place.
(3) production of test strips
4 × 1 cm(long of clip × wide) plastic-substrates, carve the hole of 0.5 × 0.5 cm in black basal region
Nitrocellulose filter (NC film) is attached to the hole region back side by region, and melamine molecular engram made from step (1) is poly-
Close object to be added dropwise in NC film surface, be dried under room temperature after being evenly distributed, then, toward MIP on that 15 mL mass percents are added dropwise is dense
The ethyl alcohol that degree is 25% is eluted 2 minutes, is eluted with secondary water, to remove the melamine in melamine molecular engram polymer
It with partial adsorbates object, dries, so that the melamine molecular engram test strips for remaining with specific recognition hole be prepared, keeps away
Light saves.
(4) preparation of testing sample solution: precision pipettes 1.0 mL milk sample to be checked in 10 mL color-comparison tubes,
With the NaAc_HAc buffer solution constant volume of 0.1 pH=5 mol/L, as sample sets；1.0 mL are pipetted with batch ox in parallel
0.5 mL 1.0 × 10 is added into 10 mL color-comparison tubes in milk sample-4 Mol/L melamine standard solution, with 0.1
The sodium acetate buffer constant volume of pH=5 mol/L is as mark-on group, and every group of parallel determination is three times.
(5) melamine molecular engram test strips made from step (3) are taken, 20 μ L GOD- are added dropwise in its reaction zone
MEL incubation 5 minutes, is eluted with 10 mL secondary waters, washes off the GOD-MEL that do not hatch up and other impurity.
(6) sample to be tested of 20 μ L step (4) sample sets is added drop-wise in the test strips after step (5) hatching, reaction 3
Minute, it is rinsed with secondary water.
(7) the dyestuff bottom liquid of 20 μ L is added dropwise in the test strips after competition (is 3 × 10 comprising concentration-4Mol/L fluorescence peach
Red, concentration is 0.02g/mL glucose, the HAc-NaAc buffer that concentration is 0.1 mol/L), it carries out chromogenic reaction 3 minutes.
(8) fluorescence cuvette is taken, it is strong that the test strips after chromogenic reaction are placed in progress fluorescence in RF5301 fluophotometer
Degree detection, which uses maximum excitation wavelength for 515 nm, and the slit width of EX and EM are respectively 1.5 nm, 3.0 nm, into
The detection of row fluorescence intensity, and with the naked eye color simply can be judged and taken pictures with ultraviolet lamp when semi-quantitative analysis,
It is compared with fluorescence standard colorimetric card.
(9) mark-on sample sets measuring method is same as above, and acquisition fluorescence data carries out recovery of standard addition experiment.
Embodiment 2:
(1) preparation of melamine molecular engram polymer
0.003 g N, N- methylene-bisacrylamide is weighed, it is 3 × 10 that 20 mL concentration, which are added,-4 The melamine of mol/L
Amine (MEL) solution and 5mL α-methacrylic acid, ultrasound 10 minutes after standing overnight, are added 0.02g ammonium persulfate and cause, instead
It answers 5 minutes, obtains melamine molecular engram polymer (MIP), stored in refrigerator, it is spare.
(2) periodate oxidation method prepares glucose oxidase label melamine (GOD-MEL)
It weighs 5 mg glucose oxidases (GOD) and is dissolved in 1 mL secondary water, it is 0.1 mol/L's that 0.2 mL concentration, which is added,
NaIO4Solution is protected from light stirring 20 minutes at room temperature；Above-mentioned solution is fitted into bag filter (MW4000), to 1 mmol/L pH=
4.4 HAC-NaAC buffer dialysis, 4 DEG C overnight, and 20 mL, 0.2 carbonate buffer solution of mol/L pH=9.5 is added, makes
The pH of GOD is increased to 9.5；Take another 10 mL colorimetric cylinder, be added 10 mg melamines and 1 mL, 0.01 mol/L pH=
9.5 carbonate buffer solution is mixed with above-mentioned GOD, and room temperature is protected from light stirring 2 hours；It is 4 that 0.1 mL concentration is added into mixed liquor
The NaBH of mg/mL4It mixes, 4 DEG C are placed 2 hours；Above-mentioned solution is fitted into bag filter, to 0.15 pH=7.4 mol/L
PBS buffer solution dialysis, 4 DEG C overnight；After the completion of dialysis, marker is distributed into 1 mL colorimetric cylinder, 4 DEG C are kept in dark place.
(3) production of test strips
4 × 1 cm(long of clip × wide) plastic-substrates, carve the hole of 0.5 × 0.5 cm in black basal region
Nitrocellulose filter (NC film) is attached to the hole region back side by region, and melamine molecular engram made from step (1) is poly-
Close object to be added dropwise in NC film surface, be dried under room temperature after being evenly distributed, then, toward MIP on that 20 mL mass percents are added dropwise is dense
The ethyl alcohol that degree is 25% is eluted 3 minutes, is eluted with secondary water, to remove the melamine in melamine molecular engram polymer
It with partial adsorbates object, dries, so that the melamine molecular engram test strips for remaining with specific recognition hole be prepared, keeps away
Light saves.
(4) 1.0 mL milk sample to be checked accurately is pipetted in 10 mL color-comparison tubes, with the vinegar of 0.1 pH=5 mol/L
Acid-sodium acetate buffer constant volume, as sample sets.1.0 mL are pipetted in parallel has plug colorimetric with batch milk sample to 10 mL
1.0 mL 1.0 × 10 are added in Guan Zhong-6 Mol/L melamine standard solution, the sodium acetate buffer with 0.1 pH=5 mol/L are molten
Liquid constant volume is as mark-on group, and every group of parallel determination is three times.
(5) the melamine molecular engram test strips for taking step (3) to prepare, are added dropwise 20 μ L GOD- in its reaction zone
MEL incubation 5 minutes, is eluted with 10 mL secondary waters, washes off the GOD-MEL that do not hatch up and other impurity.
(6) sample to be tested of 20 μ L step (4) sample sets is added drop-wise in the test strips after step (5) hatching, reaction 3
Minute, it is rinsed with secondary water.
(7) the dyestuff bottom liquid of 20 μ L is added dropwise in the test strips after competition (includes 3 × 10-4 Mol/L phloxine,
The HAc-NaAc buffer of 0.02 g/mL glucose, 0.1 mol/L), it carries out chromogenic reaction 3 minutes.
(8) fluorescence cuvette is taken, the test strips after chromogenic reaction are placed in RF5301 fluophotometer and detect its fluorescence
Intensity, setting maximum excitation wavelength are that the slit width of 515 nm, EX and EM are respectively that 1.5 nm, 3.0 nm carry out fluorescence intensity
Detection.It is and glimmering in addition, semi-quantitative analysis with the naked eye can simply judge color and be taken pictures with ultraviolet lamp
Light standard colorimetric card, which is compared, can be completed.
(9) mark-on sample sets measuring method is same as above, and acquisition fluorescence data carries out recovery of standard addition experiment.
Embodiment 3:
(1) preparation of melamine molecular engram polymer
0.0032 g N, N- methylene-bisacrylamide is weighed, it is 3 × 10 that 20 mL concentration, which are added,-4 The trimerization of mol/L
Cyanamide (MEL) solution and 7mL α-methacrylic acid, ultrasound 10 minutes after standing overnight, are added 0.03g ammonium persulfate and cause,
Reaction 7 minutes, obtains melamine molecular engram polymer (MIP), stores in refrigerator, spare.
(2) periodate oxidation method prepares glucose oxidase label melamine (GOD-MEL)
It weighs 6 mg glucose oxidases (GOD) and is dissolved in 1 mL secondary water, it is 0.1 mol/L's that 0.2 mL concentration, which is added,
NaIO4Solution is protected from light stirring 20 minutes at room temperature；Above-mentioned solution is fitted into bag filter (MW4000), to 1 mmol/L pH=
4.4 HAC-NaAC buffer dialysis, 4 DEG C overnight, and 20 mL, 0.2 carbonate buffer solution of mol/L pH=9.5 is added, makes
The pH of GOD is increased to 9.5；Take another 10 mL colorimetric cylinder, be added 12 mg melamines and 1 mL, 0.01 mol/L pH=
9.5 carbonate buffer solution is mixed with above-mentioned GOD, and room temperature is protected from light stirring 2 hours；It is 4 that 0.1 mL concentration is added into mixed liquor
The NaBH of mg/mL4It mixes, 4 DEG C are placed 2 hours；Above-mentioned solution is fitted into bag filter, to 0.15 pH=7.4 mol/L
PBS buffer solution dialysis, 4 DEG C overnight；After the completion of dialysis, marker is distributed into 1 mL colorimetric cylinder, 4 DEG C are kept in dark place.
(3) production of test strips
4 × 1 cm(long of clip × wide) plastic-substrates, carve the hole of 0.5 × 0.5 cm in black basal region
Nitrocellulose filter (NC film) is attached to the hole region back side by region, and melamine molecular engram made from step (1) is poly-
Close object to be added dropwise in NC film surface, be dried under room temperature after being evenly distributed, then, toward MIP on that 25 mL mass percents are added dropwise is dense
The ethyl alcohol that degree is 25% is eluted 4 minutes, is eluted with secondary water, to remove the melamine in melamine molecular engram polymer
It with partial adsorbates object, dries, so that the melamine molecular engram test strips for remaining with specific recognition hole be prepared, keeps away
Light saves.
(4) preparation of testing sample solution: precision pipettes 1.0 mL milk sample to be checked in 10 mL color-comparison tubes,
With the NaAc_HAc buffer solution constant volume of pH=5 0.1mol/L, as sample sets.1.0 mL are pipetted with batch milk in parallel
0.4 mL 1.0 × 10 is added into 10 mL color-comparison tubes in sample-6 Mol/L melamine standard solution, with 0.1 mol/L
The sodium acetate buffer constant volume of pH=5 is as mark-on group, and every group of parallel determination is three times.
(5) melamine molecular engram test strips made from step (3) are taken, 20 μ L glucoses are added dropwise in its reaction zone
Oxidase label melamine solution incubation 5 minutes, is eluted with 10 mL secondary waters, washes off the GOD- that do not hatch up
MEL and other impurity.
(6) sample to be tested of 20 μ L step (4) sample sets is added drop-wise in the test strips after step (5) hatching, reaction 3
Minute, it is rinsed with secondary water.
(7) the dyestuff bottom liquid of 20 μ L is added dropwise in the test strips after competition (includes 3 × 10-4 Mol/L phloxine,
The HAc-NaAc buffer of 0.02g/mL glucose, 0.1 mol/L), it carries out chromogenic reaction 3 minutes.
(8) fluorescence cuvette is taken, it is strong that the test strips after chromogenic reaction are placed in progress fluorescence in RF5301 fluophotometer
Degree detection, the system use maximum excitation wavelength for 515 nm, and the slit width of EX and EM are respectively that 1.5 nm, 3.0 nm are carried out
Fluorescence intensity detection, and with the naked eye color simply can be judged and taken pictures with ultraviolet lamp when semi-quantitative analysis, with
Fluorescence standard colorimetric card is compared.
(9) mark-on sample sets measuring method is same as above, and acquisition fluorescence data carries out recovery of standard addition experiment.
According to above-mentioned detection method and calculation formula, addition melamine, mark-on reclaims are not detected in three batches of milk samples
For rate in 91.2% ~ 112.05% range, relative standard deviation is 2.3% ~ 2.7%.The measuring method of melamine is returned
Yield and relative standard deviation are shown in Table 1.
Claims (1)
1. a kind of method of quickly detection melamine, it is characterised in that specific steps are as follows:
(1) preparation of melamine molecular engram polymer
0.0020 ~ 0.0032 g N, N- methylene-bisacrylamide is weighed, it is 3 × 10 that 20 mL concentration, which are added,-4 Mol/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, obtains melamine molecular engram polymer, store in refrigerator, spare；
(2) periodate oxidation method prepares glucose oxidase label melamine
It weighs 4 ~ 6 mg glucose oxidases and is dissolved in 1 mL secondary water, the NaIO that 0.2 mL concentration is 0.1 mol/L is added4It is molten
Liquid is protected from light stirring 20 minutes at room temperature；Above-mentioned solution is fitted into bag filter, is 1 mmol/L, pH's=4.4 to concentration
The dialysis of HAC-NaAC buffer, 4 DEG C overnight；It is 0.2 carbonate buffer solution of mol/L pH=9.5 that 20 mL concentration, which are added, is made
The pH of glucose oxidase is increased to 9.0 ~ 9.5；Another 10 mL colorimetric cylinder is taken, 8 ~ 12 mg melamines and 1 are added
ML concentration is the carbonate buffer solution of 0.01 pH=9.5 mol/L, is mixed with above-mentioned glucose oxidase, room temperature, which is protected from light, to be stirred
It mixes 2 hours；The NaBH that 0.1 mL concentration is 4 mg/mL is added into mixed liquor4It mixes, 4 DEG C are placed 2 hours；It will be above-mentioned molten
Liquid is fitted into bag filter, is dialysed to the PBS buffer solution that concentration is 0.15 mol/L, pH=7.4, and 4 DEG C overnight；Dialysis is completed
Afterwards, marker is distributed into 1 mL colorimetric cylinder, 4 DEG C are kept in dark place；
(3) production of test strips
Clip length × width is the plastic-substrates of 4 × 1 cm, carves the hole area of 0.5 × 0.5 cm in black basal region
Nitrocellulose filter is attached to the hole region back side by domain, and melamine molecular engram polymer made from step (1) is added dropwise
In nitrocellulose film surface, dried under room temperature after being evenly distributed；Then, toward being added dropwise 15 on melamine imprinted polymer
The ethyl alcohol that ~ 25 mL mass percent concentrations are 25% is eluted 2 ~ 4 minutes, is eluted with secondary water, to remove melamine point
Melamine and partial adsorbates object in sub- imprinted polymer, dry, and remain with specific recognition hole to be prepared
Melamine molecular engram test strips, are kept in dark place；
(4) detection method
Melamine molecular engram test strips one is taken made from step (3), and the grape that 15 ~ 25 μ L have been marked is glycoxidative
Enzyme label melamine solution is added drop-wise in the molecular engram test strips for having removed template molecule, and incubation 5 minutes, with two
The glucose oxidase label melamine and other impurity that do not hatch up are washed off in secondary water elution；Competition process is not
With what is carried out in the sample of concentration melamine, 15 ~ 25 μ L testing sample solutions are added dropwise in the test strips to after hatching, competition
Reaction 3 minutes, is rinsed with secondary water；It is 3 × 10 that 15 ~ 25 μ L, which are added dropwise, comprising concentration in test strips after competition-4 Mol/L's
Phloxine, concentration are the dyestuff bottom liquid of the glucose of 0.02 g/mL, the HAc-NaAc buffer that concentration is 0.1 mol/L, into
Row chromogenic reaction；The maximum excitation wavelength for adjusting fluophotometer is that 515 nm, exciting light i.e. EX slit width are 1.5 nm, send out
Penetrating light i.e. EM slit width is 3.0 nm, and the test strips after chromogenic reaction are placed in progress fluorescence intensity inspection in fluophotometer
It surveys, realizes quantitative analysis；In addition, the on-line monitoring of batch samples can be completed by semi-quantitative analysis, i.e., it is with the naked eye right
Color is simply judged with optical colorimetry or carries out comparison of taking pictures with ultraviolet lamp；
(5) drafting of working curve
Melamine molecular engram test strips made from step (3) are taken, it is molten by melamine standard of the step (4) to various concentration
Liquid carries out fluorescence detection, records fluorescence intensity, draws working curve；By the logarithmic regressions analysis to working curve it is found that examination
The fluorescence intensity difference and 1 × 10 of paper slip-8 mol/L ~ 1×10-4The logarithm of mol/L melamine concentration is in a linear relationship, line
Property regression equation isΔI F = 61.19lgC + 572.83, linearly dependent coefficient reaches 0.9944；
(6) in milk sample content of melamine measurement
300 ~ 600 commercially available milk samples of μ L are pipetted, it is molten with the acetic acid sodium acetate buffer that concentration is 0.1 mol/L, pH=5
Liquid is settled to 10 mL, as sample to be tested；Melamine molecular engram test strips one is taken made from step (3), will be marked
Glucose oxidase label 20 μ L of melamine solution be added drop-wise in the molecular engram test strips for having removed template molecule,
It incubation 5 minutes, is eluted with secondary water, washes off the glucose oxidase label melamine that do not hatch up and others are miscellaneous
Matter；20 μ L testing sample solutions are added dropwise in the test strips to after hatching, reacts 3 minutes, is rinsed with secondary water；After competition
It is 3 × 10 that 20 μ L, which are added dropwise, comprising concentration in test strips-4 The 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 chromogenic reaction are placed in glimmering
Fluorescence intensity detection is carried out in light photometer, and the concentration of melamine in sample is calculated according to working curve.
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| CN103175818A (en) * | 2013-03-19 | 2013-06-26 | 桂林理工大学 | Method for detecting melamine |
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| CN105241944A (en) * | 2015-09-25 | 2016-01-13 | 浙江大学 | Method for electrochemical detection of antibiotics in milk based on vertical and ordered micelle enrichment |
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| CN101559352A (en) * | 2009-05-15 | 2009-10-21 | 华南农业大学 | Molecularly imprinted polymers (MIPs) for inspecting melamine and preparation method thereof |
| WO2013105090A1 (en) * | 2012-01-11 | 2013-07-18 | Aptateck Bio Ltd. | A versatile lateral flow strip device |
| CN103175818A (en) * | 2013-03-19 | 2013-06-26 | 桂林理工大学 | Method for detecting melamine |
| CN104914080A (en) * | 2015-05-14 | 2015-09-16 | 桂林理工大学 | Rapid detection method of clenbuterol |
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