CN104359897A - Method for rapidly detecting melamine based on nanogold chemiluminiscence - Google Patents
Method for rapidly detecting melamine based on nanogold chemiluminiscence Download PDFInfo
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- CN104359897A CN104359897A CN201410576585.1A CN201410576585A CN104359897A CN 104359897 A CN104359897 A CN 104359897A CN 201410576585 A CN201410576585 A CN 201410576585A CN 104359897 A CN104359897 A CN 104359897A
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- chemiluminescence
- gold
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Abstract
The invention discloses a method for rapidly detecting melamine based on nanogold chemiluminiscence. The method comprises the following steps: step 1), transferring a nanogold solution and a solution containing melamine into a centrifuge tube, fully mixing, and acting for five minutes to promote nanogold to reach an optimal agglomeration state; step 2), transferring a mixed solution of the nanogold solution and the melamine solution into a chemiluminiscence bath, filling a Luminol-hydrogen peroxide chemiluminiscence reagent in a static injection mode, measuring and recording the chemiluminiscence strength through an IFFL-D flow injection chemiluminescent analyzer, and judging according to measuring results. The detection limit reaches 8.6*10-14g/mL, and the method reaches the sensitivity of the existing solid-phase extraction and chemiluminiscence analysis method and is simpler and more convenient than the existing solid-phase extraction and chemiluminiscence analysis method. The analysis and test time is shortened within ten minutes. The conditions of the whole operation experiment are mild and are finished in a homogeneous system, thereby facilitating automatic analysis operation.
Description
Technical field
The invention belongs to technical field of chemical detection, specifically, the present invention relates to a kind of method detecting melamine based on nm of gold chemiluminescence fast.
Background technology
Melamine (melamine) chemical name is 2,4,6-tri-amido-1,3,5-triazine (2,4,6-triamino-1,3,5-triazine) or 1,3,5-triazine-2,4,6-triamine (1,3,5-triazine-2,4,6-triamino), be a kind of triazines nitrogen heterocyclic ring organic compound, be commonly called as melamine, extract of protein.As a kind of important azacyclo-Organic Chemicals, main application is and aldehyde condensation, produces plastics.Because its form exactly likes albumen powder, free from extraneous odour, nitrogen content is up to 66%, and far above the nitrogen content of usual protein, chronic toxicity is not easy to be found.Add the apparent upper protein massfraction raising that these product can cause Kjeldahl's method to record, product quality " qualified ", significantly reduces production cost simultaneously, is therefore often added in dairy products, feed as protein replacement by lawless person.The toxicity of melamine own is less, takes in and may have an impact to kidney and bladder for a long time or repeatedly, cause calculus to produce.In view of the artificial interpolation of melamine can work the mischief to consumer, relevant department of China has issued the bulletin of melamine Limited Doses.Therefore, the effective safety detected for food especially dairy products is carried out to melamine and there is certain meaning.
Be usually used in the detection method of melamine at present both at home and abroad, mainly contain high performance liquid chromatography (HPLC), Liquid Chromatography-Mass Spectrometry, immunoassay, capillary electrophoresis, gas chromatography mass spectrometry method, fluorimetry etc.These methods and each have their own advantage of technology and usable range.In these analytical approachs, HPLC method is the most frequently used detection method, has efficient, quick, highly sensitive, recall rate high; But because pre-treatment is more complicated, required instrument is costly, complex operation, time-consuming, agents useful for same toxicity is comparatively large, contaminated environment, and has certain technical requirement to experiment operator, is unsuitable for the quick detection of melamine.Although gas chromatography mass spectrometry and LC-MS technology are detection methods more advanced at present, its apparatus expensive, reagent consumption is large, and testing cost is high, only can use as confirmation method in part Experiment room, be not suitable for widely using.Capillary electrophoresis degree of separation is high, good separating effect, and agents useful for same amount is little, inexpensive pollution-free, but insufficient sensitivity is high.Although near infrared absorption method selectivity is good, can realize on-line checkingi, but equipment price is expensive, needs constantly to calibrate, and the interference in sample substrate is near-infrared analysis precision and reproducible one large obstacle.Immunoassay is highly sensitive, is applicable to the rapid screening of a large amount of sample, but complex operation, time-consuming.Colourimetry is convenient and swift, inexpensive easy to operate, but is only applicable to screen when content of melamine is large, cannot carry out trace detection, and when the large time error of determinand alkalescence is also larger.Although Raman spectroscopy and nuclear magnetic resonance method can meet quick easy requirement, instrument itself costly.Therefore, develop easy, the quick and practical analytical approach for melamine detection and seem necessary.
For the relative deficiency that prior art exists, the present invention is proposed.
Summary of the invention
The object of the invention is to solve the limitation detecting melamine analytical approach at present and exist: comprise the instrument using Large expensive, the demand that analysis cost is high, sensitivity can not meet each side; Troublesome poeration and higher to the requirement of analyst; Analyze minute longer.Utilize melamine can strengthen luminol-hydrogen peroxide-nm of gold system chemiluminescence signal, set up a kind of brand-new simple and sensitive measures melamine method based on nano gold catalysis chemiluminescence analysis.
Technical scheme provided by the invention is: a kind of method detecting melamine based on nm of gold chemiluminescence fast, comprises the following steps:
Step 1), pipette nano-Au solution and containing the solution of melamine in a centrifuge tube, fully mix, act on the reunion state making nm of gold reach the best for 5 minutes; The particle diameter of the nm of gold in described nano-Au solution is 2.6nm, and concentration is 5.0 × 10
-7mol/L.The preparation process of described 2.6nm nano-Au solution is: under the condition of room temperature, strong stirring, by the HAuCl of 100mL
4(0.01%w/w) 1% citric acid three sodium solution of aqueous solution, 1mL and the 0.075%NaBH of 1mL
4/ 1% citric acid three sodium solution mixes completely, and after mixing, solution stirs 30 minutes, after complete reaction, stand-by in 4 DEG C of Refrigerator stores.Described Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent selects the concentration of luminol to be 4 × 10
-5mol/L, concentration of hydrogen peroxide is 4 × 10
-2mol/L, the volume ratio of luminol and hydrogen peroxide is 2:1.The pH value of described Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent is 12.0.
Step 2), pipette the mixed solution of above-mentioned nano-Au solution and melamine solution in chemiluminescence pond, the mode of static state feedback is adopted to inject Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent, measured by IFFL-D Flow Injection Analysis/Chemiluminescence instrument and record its chemiluminescence intensity, judging according to measurement result.
Other technical scheme provided by the invention is: by step 1) described in Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent luminol-potassium metaperiodate chemical illuminating reagent replace.
The invention has the beneficial effects as follows:
Chemiluminescence analysis because of have highly sensitive, the range of linearity is wide, reagent is cheap and instrumentation is simple etc., and advantage has been widely used in Pharmaceutical Analysis.And chemiluminescence analysis detects the research report of melamine seldom.In recent years, along with the fast development of nano science, nano material is used in chemiluminescence bioanalysis with various role because of its physicochemical property such as distinctive optics and electricity, has started the new neighborhood that nano particle chemistry luminous organism is analyzed.Once becoming the focus of research.Unfortunately, the research based on nano gold catalysis chemiluminescence analysis mensuration melamine have not been reported.Our research finds that melamine can strengthen the chemiluminescence signal of luminol-hydrogen peroxide-nm of gold system, establishes the analytical approach that a kind of nano gold catalysis chemiluminescence analysis measures melamine, the method simple and sensitive, fast based on this.Whole analysis mensuration will complete within 10 minutes.
The method detecting melamine based on nano gold catalysis chemiluminescence analysis that the present invention sets up, solves the problem that existing detection method runs into, has the following advantages: (1) detection limit reaches 8.6 × 10
– 14g/mL, reaches the sensitivity of current Solid-Phase Extraction-chemiluminometry, and than many simply and easily; (2) the analytical test time shorten of the method is within 10 minutes; (3) condition of whole operation experiments is gentleer, and completes in the system of a homogeneous phase, is conducive to the analysis operation of robotization.
Accompanying drawing explanation
When considered in conjunction with the accompanying drawings, by referring to detailed description below, more completely can understand the present invention better and easily learn wherein many adjoint advantages, but accompanying drawing described herein is used to provide a further understanding of the present invention, form a part of the present invention, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention, wherein:
The optimization schematic diagram of Fig. 1 nm of gold concentration;
The optimization schematic diagram of Fig. 2 luminol concentration;
The optimization schematic diagram of Fig. 3 concentration of hydrogen peroxide;
The selection schematic diagram of Fig. 4 system pH;
The sensitivity analysis schematic diagram of Fig. 5 chemiluminescence detection melamine.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, and below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Used key instrument equipment and reagent:
IFFL-D Flow Injection Analysis/Chemiluminescence instrument and support equipment (Xi'an Rui Mai Analytical Instrument Co., Ltd) thereof, 5840R desk type high speed Large Copacity hydro-extractor (Eppendorf Ltd. Germany), TU-1901 type spectrophotometry instrument (Beijing Pu Xi all purpose instrument company limited), transmission electron microscope (Hitachi, Ltd, specifications and models: H-600).
Gold chloride (HAuCl
44H
2o, AR, Au content >47.8%, Chemical Reagent Co., Ltd., Sinopharm Group); Gold chloride solid is dissolved in secondary deionized water and prepares gold chloride stock solution, be put in 4 DEG C of refrigerators and preserve.4.43g luminol pressed powder to be dissolved in 20mL0.10M NaOH solution and to be diluted to 1L, preparing 2.5 × 10
– 2m luminol stock solution.Keep in Dark Place a week to guarantee the stable of reagent properties before use.The working solution of luminol is obtained by the dilution of stock solution.H
2o
2working solution when each use from 30% (w/w) H
2o
2(Solution on Chemical Reagents in Shanghai company) dilution obtains.Take melamine (Beijing Xing Fu fine chemistry research institute) 0.0050g, after being dissolved in water, constant volume, in 100ml volumetric flask, configures 1 × 10
-4g/ml melamine storing solution.Experimental water is secondary deionized water.
The optimization of experiment condition:
Select the nm of gold of 2.6nm in experiment, examine the impact of the concentration of gold.As Fig. 1, the concentration of gold is larger, and the luminous intensity of system is also larger, and when gold concentration is greater than 5.0 × 10
-7during mol/L, the chemiluminescence intensity of system weakens to some extent.The present invention selects the particle diameter of nm of gold to be 2.6nm, and concentration is 5.0 × 10
-7mol/L.
Investigated the impact of chemical illuminating reagent, as Fig. 2 and 3, find the increase along with luminol and concentration of hydrogen peroxide, the luminous intensity of system is larger.Consider the consumption of chemiluminescence intensity and reagent, the concentration selecting luminol is 4 × 10
-5mol/L, concentration of hydrogen peroxide is 4 × 10
-2mol/L.
Investigate the impact of system pH, as Fig. 4, when the pH value of system is less than 12, along with the increase chemiluminescence intensity of pH value increases; And when the pH value of system is greater than 12, along with the increase chemiluminescence intensity of pH value weakens.The pH value of the present invention's system used is optimized for 12.0.
The preparation of 2.6nm nm of gold:
Glassware used in preparation process all uses chloroazotic acid to clean (1:3HNO
3– HCl), then use secondary deionized water cleaning down clean, dry for standby.The preparation method of 2.6nm nm of gold is sodium borohydride reduction.Preparation process is summarized as follows: under the condition of room temperature, strong stirring, by the HAuCl of 100mL
4(0.01%w/w) 1% citric acid three sodium solution of aqueous solution, 1mL and the 0.075%NaBH of 1mL
4/ 1% citric acid three sodium solution mixes completely.After mixing, solution stirs 30 minutes, after complete reaction, stand-by in 4 DEG C of Refrigerator stores.
Static state feedback chemiluminescence analysis measures melamine:
The chemiluminescence analysis that the present invention sets up measures the method for melamine, adopts the mode of static state feedback to carry out.Whole process is divided into two steps: the interaction of melamine and nm of gold and the mensuration of chemiluminescence analysis, and these two steps are completely independently.Concrete mensuration process is as follows: (1) pipettes the solution of nm of gold and melamine in a centrifuge tube, fully mixes, and acts on the reunion state making nm of gold reach best for 5 minutes; (2) pipette above-mentioned nm of gold/melamine solution 100 μ L in chemiluminescence pond (40 × 14mm quartz ampoule), inject 200 μ L Shandong rice promise – hydrogen peroxide chemistry luminescence reagents (4 × 10
-5m luminol and 4 × 10
-2the volume ratio of M hydrogen peroxide is 2:1), measured by IFFL-D Flow Injection Analysis/Chemiluminescence instrument and record its chemiluminescence intensity.
The chemiluminescent analytical performance of melamine:
Under the experiment condition of above-mentioned optimization, achieve the mensuration to melamine chemiluminescence analysis.Result shows, along with the increase of melamine concentration, chemiluminescence signal strengthens gradually.And 3.8 × 10
-12~ 2.6 × 10
-10in the concentration range of g/mL, the concentration of melamine becomes good correlativity with the intensity of chemiluminescence signal, and as shown in Figure 5, it detects and is limited to 8.6 × 10 linearity curve
– 14g/mL (S/N=3).And to 1.6 × 10
-10the melamine replicate determination of g/mL 11 times, its relative standard deviation is 3.2%.Experimental result shows that the method detection limit is low, and precision is good.The sensitivity of more current only chemiluminescence analysis method wants high a lot, has reached the sensitivity of Solid-Phase Extraction-chemiluminometry, and than many simply and easily.
Interference measurement
In 8.6 × 10
– 10in g/mL melamine solution, add the Zn of 1000 times respectively
2+, Ca
2+, Mg
2+, NO
3 -, CO
3 2-, P0
4 3-and glucose, the Al of 100 times
3+, Na
+, Cl
-, SO
4 2-, urea and citric acid, the Cu of 10 times
2+, Ba
2+etc. carrying out interference experiment, result shows all not interference measurements.
The result of sample analysis:
By National Standard Method processing sample.Accurately pipette 1mL fresh milk sample, add acetonitrile (pH=2.0) solution of 5mLHCl acidifying, ultrasonic 15 minutes, make protein precipitation, then again with 8000r/min centrifugal 10 minutes, transfer merges supernatant, adds 20mL normal hexane, vibrate, leave standstill, discard upper organic phase in above-mentioned clear liquid, by subnatant evaporated under reduced pressure, water-soluble, use filtering with microporous membrane, uses water constant volume, and vortex mixed obtains clear liquid after 1 minute for subsequent use.
Adopt standard addition method to carry out the experiment of the recovery, the results are shown in table 1.Experimental result shows, the method that the present invention sets up has good accuracy and precision, the recovery is respectively 97.79% ~ 102.13%, RSD is 1.8% ~ 4.6%, and sample pre-treatments is simple, analysis time is short, does not need to use large-scale instrument, can be used for the quick detection of melamine in milk sample.
The measurement result (n=5) of melamine in table 1 milk
Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent in the present invention can use luminol-potassium metaperiodate chemical illuminating reagent to replace, and all the other are identical with the present invention, also can reach identical effect, repeat no more herein.The explanation of above example just understands core concept of the present invention for helping; Meanwhile, for one of ordinary skill in the art, according to thought of the present invention, all will change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention.
Claims (5)
1. detect a method for melamine based on nm of gold chemiluminescence fast, it is characterized in that, comprise the following steps:
Step 1), pipette nano-Au solution and containing the solution of melamine in a centrifuge tube, fully mix, act on the reunion state making nm of gold reach the best for 5 minutes;
Step 2), pipette the mixed solution of above-mentioned nano-Au solution and melamine solution in chemiluminescence pond, the mode of static state feedback is adopted to inject Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent, measured by IFFL-D Flow Injection Analysis/Chemiluminescence instrument and record its chemiluminescence intensity, judging according to measurement result.
2. the method detecting melamine based on nm of gold chemiluminescence fast according to claim 1, is characterized in that, the particle diameter of the nm of gold in described nano-Au solution is 2.6nm, and concentration is 5.0 × 10
-7mol/L.
3. the method detecting melamine based on nm of gold chemiluminescence fast according to claim 1, is characterized in that, described Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent selects the concentration of luminol to be 4 × 10
-5mol/L, concentration of hydrogen peroxide is 4 × 10
-2mol/L, the volume ratio of luminol and hydrogen peroxide is 2:1.
4. the method detecting melamine based on nm of gold chemiluminescence fast according to claim 1, is characterized in that, the pH value of described Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent is 12.0.
5. the method detecting melamine based on nm of gold chemiluminescence fast according to claim 1, is characterized in that, described Lu meter Nuo – hydrogen peroxide chemistry luminescence reagent luminol-potassium metaperiodate chemical illuminating reagent replaces.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111308094A (en) * | 2020-03-03 | 2020-06-19 | 浙江卓运生物科技有限公司 | Homogeneous method troponin chemiluminescence detection reagent and preparation method thereof |
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Cited By (2)
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Application publication date: 20150218 |