CN103115954A - Method for fast measuring melamine in food by anthraquinone-2-sodium sulfonate sensor - Google Patents
Method for fast measuring melamine in food by anthraquinone-2-sodium sulfonate sensor Download PDFInfo
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- CN103115954A CN103115954A CN2013100362332A CN201310036233A CN103115954A CN 103115954 A CN103115954 A CN 103115954A CN 2013100362332 A CN2013100362332 A CN 2013100362332A CN 201310036233 A CN201310036233 A CN 201310036233A CN 103115954 A CN103115954 A CN 103115954A
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Abstract
The invention belongs to the field of food detection, and particularly relates to a method for fast measuring melamine in a sample by an anthraquinone-2-sodium sulfonate sensor. The method comprises the following steps of: preparing a standard melamine solution; drawing a standard curve; preparing a sample to be measured; and detecting the sample. The method disclosed by the invention can be used for detecting whether melamine is contained in the food sample or not and the melamine content, has the advantages of real-time and fast detection, can complete the integral detection process within 30 minutes, achieve the minimal detection limit at 5.00*10<-9>M (0.6 microgram/millilitre) and bring about great convenience for the supervision and control of market food safety, and has the advantages of low detection cost, real-time and fast detection on food in market and production, and the like.
Description
Technical field
The invention belongs to the food inspection field, be specifically related to a kind of method that adopts melamine in anthraquinone-2-sodium sensor Fast Measurement sample.
Background technology
For many years, food security is the topic that people are concerned about always, contain melamine from the pet food of China's export in 2006, investigated and prosecuted by the U.S., contain the outburst of melamine event to China Partial baby formula milk powder in 2009, make people push the peak to the concern of melamine in food and feed.
Melamine is a kind of triazines nitrogen heterocyclic ring organic compound, is usually used in timber processing, plastics, coating, papermaking, weaving, leather, the industry such as electric, medical.Melamine has certain renal toxicity, can cause death when serious.Because the average nitrogen content of protein is only 16%, and the nitrogen content of melamine is 66% left and right, therefore melamine often is used as food additives (especially baby formula milk powder and feed for pet) by illegal producer, to improve the Protein Index (with Kjeldahl nitrogen determination) in food inspection.
The traditional detection method of melamine has gravimetric method, sublimed method.But these two kinds of method complex operations, analysis time is oversize.The method that both at home and abroad melamine in milk is detected at present has a lot of relevant research and reports, mainly contains gas chromatography-mass spectrography, high performance liquid chromatography, liquid chromatography-mass spectrography etc.Though the content of melamine in the measurement sample that these detection methods can be accurate, sensitive, but the analyst and the complicated preprocessing process that need expensive large-scale instrument, specialty, and need to consume a large amount of organic solvents, be difficult to realize production run and product are carried out real-time Site Detection, also be unsuitable for enforcing the law and the field quick detection of superintendent office.Therefore stable, reliable, high sensitivity, low detectability of research and the method for quick that is applicable to Real-Time Monitoring have become the trend of domestic and international research.
Chemical sensor due to have fast, high sensitivity, low detectability and the advantage such as easy and simple to handle, drawn a domestic and international numerous scholar concern and interest.And at present a large amount of explorations has been carried out in the development of toxic chemical chemical sensor, some be successfully applied to quality testing department and environment superintendent office in real time, fast detecting.But the chemical sensor that is used for the melamine fast detecting rarely has report.
Summary of the invention
In order to solve above-mentioned technical matters, the invention provides a kind of method that adopts melamine in anthraquinone-2-sodium sensor Fast Measurement sample.
In employing anthraquinone-2-sodium sensor Fast Measurement sample of the present invention, the method for melamine is to solve above technical matters by following technical scheme:
Adopt the method for melamine in anthraquinone-2-sodium sensor Fast Measurement sample, comprise following step:
A. prepare the melamine standard solution: take the melamine standard items, in the 100mL volumetric flask, be mixed with the standard solution that concentration is 1 mol/L with methyl alcohol dissolving and constant volume, be placed under 4 ℃ and keep in Dark Place;
Preparation anthraquinone-2-sulfonic acid sodium solution: accurately take 0.6205 gram anthraquinone-2-sodium, be settled to 200mL with the phosphate buffer dissolving that pH is 4.0, concentration is 0.1mol/L, being mixed with concentration is the anthraquinone-2-sodium stock solution of 0.01mol/L, is diluted to corresponding concentration with damping fluid again during use;
B. drawing standard curve: add successively the melamine standard solution in 6 100mL volumetric flasks, and with 1 * 10
-4Mol/L anthraquinone-2-sulfonic acid sodium solution constant volume makes that in volumetric flask, the concentration of melamine is followed successively by 0 mol/L, 1 * 10
-9Mol/L, 1 * 10
-8Mol/L, 1 * 10
-7Mol/L, 1 * 10
-5Mol/L, 1 * 10
-4Mol/L uses Differential Pulse Voltammetry oxygen determination galvanic current, with the linear relationship drawing standard curve between oxidation peak current reduction value and melamine concentration after three minutes;
C. testing sample preparation
Solid sample: sample is pulverized, accurately take 5 grams, add 10mL300 g/L trichloroacetic acid solution, eddy current mixing 5min with centrifugal 5 min of 3500 r/min, then transfers to supernatant in another centrifuge tube, transfer pH to 4.0 with phosphoric acid solution, standing 1 min, then with the 3500 centrifugal 3min of r/min, take out supernatant standby;
Fluid sample: the trichloroacetic acid solution that adds 1.2 mL300 g/L in the 25.0 mL testing samples, eddy current mixes 1 min, with centrifugal 5 min of 3500 r/min, then supernatant is transferred in another centrifuge tube, transfer pH to 4.0 with phosphoric acid solution, standing 1 min, then with the 3500 centrifugal 3min of r/min, take out supernatant standby;
D. the detection of sample: adding concentration in the supernatant in the step c is 1 * 10
-4The anthraquinone-2-sulfonic acid sodium solution of mol/L with Differential Pulse Voltammetry oxygen determination galvanic current, draws the melamine concentration value of testing sample to be measured after three minutes at last according to typical curve.
Anthraquinone-2-sodium (AQ) is a kind of anionic dye with quinoid structure, has good electrochemical activity.The bright anthraquinone-2-sodium of selecting of written or printed documents is molecule sensor, utilize the variation of the electrochemical response signal that causes after the combination of anthraquinone-2-sodium and melamine (MA), the electrochemical sensor that has prepared the melamine fast detecting has been set up take anthraquinone-2-sodium as the new method of electroactive sensor indirect determination without electroactive MA.Adopt method of the present invention that the content that whether contains melamine and melamine in food samples is detected, have advantages of real-time detection, fast detecting, will bring great convenience for the supervision of market food security and supervision.And the advantage such as method cost of the present invention is low, the real-time detection that can realize market and production, fast detecting.
Description of drawings
Fig. 1 is QA, the MA cyclic voltammogram on GCE; (AQ and MA concentration are respectively 1 * 10
-4Mol/L, 5 * 10
-5Mol/L; A is respectively AQ, AQ+MA, MA to c);
Fig. 2 is the reaction time of AQ and MA; In figure, AQ and MA concentration are respectively 1 * 10
-4Mol/L, 5 * 10
-5Mol/L;
Fig. 3 is the impact of pH, and a-f pH is respectively 3,4,5,6,7,8; AQ and MA concentration are respectively 1 * 10
-4Mol/L, 5 * 10
-5Mol/L;
Fig. 4 is that under different PH, peak current changes schematic diagram;
The mixed DPV figure of AQ and MA when Fig. 5 is different MA concentration; AQ concentration is 1 * 10
-4Mol/L, the concentration difference 0,1 * 10 of a-f MA
-9, 1 * 10
-8, 1 * 10
-7, 1 * 10
-5, 1 * 10
-4Mol/L;
Fig. 6 is the relation between current reduction value and MA concentration, and interior illustration is that AQ concentration is 1 * 10
-4During mol/L, the linear relationship chart between oxidation peak current reduction value and MA concentration.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further described, so that those skilled in the art more understands the present invention, but does not limit the present invention with this.
Adopt the method for melamine in anthraquinone-2-sodium sensor Fast Measurement sample, comprise following step:
A. prepare the melamine standard solution: take the melamine standard items, in the 100mL volumetric flask, be mixed with the standard solution that concentration is 1 mol/L with methyl alcohol dissolving and constant volume, be placed under 4 ℃ and keep in Dark Place;
Preparation anthraquinone-2-sulfonic acid sodium solution: accurately take 0.6205 gram AQ, be settled to 200mL with the phosphate buffer dissolving that pH is 4.0, concentration is 0.1mol/L, being mixed with concentration is the anthraquinone-2-sodium stock solution of 0.01mol/L, is diluted to corresponding concentration with damping fluid again during use;
B. drawing standard curve: add successively the melamine standard solution in 6 100mL volumetric flasks, and with 1 * 10
-4Mol/L anthraquinone-2-sulfonic acid sodium solution constant volume makes that in volumetric flask, the concentration of melamine is followed successively by 0 mol/L, 1 * 10
-9Mol/L, 1 * 10
-8Mol/L, 1 * 10
-7Mol/L, 1 * 10
-5Mol/L, 1 * 10
-4Mol/L uses Differential Pulse Voltammetry oxygen determination galvanic current, with the linear relationship drawing standard curve between oxidation peak current reduction value and melamine concentration after three minutes;
Glass-carbon electrode is used respectively 0.3 μ m, 0.05 μ m Al
2O
3Polishing on special-purpose chamois leather, then with the electrode of polishing cleaning each 5min of each ultrasonic cleaning in double distilled water and absolute ethyl alcohol respectively, clean with redistilled water, room temperature is dried standby.Prepare AQ and the MA solution of variable concentrations with the phosphate buffer solution (PBS) of 0.1mol/L, adopt three-electrode system (take glass carbon (GCE) electrode as working electrode, saturated calomel electrode (SCE) is contrast electrode, and platinum electrode is to electrode) at the electrochemical behavior of measuring AQ and MA under different condition on the CHI660 electrochemical workstation;
C. testing sample preparation: the trichloroacetic acid solution that adds 300 g/L of 1.2mL in the 25.0mL testing sample, stir 1 min, with centrifugal 5 min of 3500 r/min, then supernatant is transferred in another centrifuge tube, transfer pH to 4.0 with phosphoric acid solution, standing 1 min, then with the 3500 centrifugal 3min of r/min, take out supernatant standby;
D. the detection of sample: adding concentration in the supernatant in the step c is 1 * 10
-4The anthraquinone-2-sulfonic acid sodium solution of mol/L with Differential Pulse Voltammetry oxygen determination galvanic current, draws the melamine concentration value of testing sample to be measured after three minutes at last according to typical curve.
Take anthraquinone-2-sodium as electrochemical sensor, measured the electrochemical behavior of AQ in the phosphate buffered solution of 0.1 mol/L.Cyclic voltammetric the results are shown in accompanying drawing 1, when showing pH=4, AQ-0.3～-have between 0.4V a pair of significantly will definitely contrary redox the peak, and this moment, MA there is no the peak.After adding MA, the oxidation-reduction potential of AQ is substantially constant, and peak current significantly reduces, and shows AQ after MA is combined, and the reaction of AQ on glass-carbon electrode is suppressed.
Melamine is with after anthraquinone-2-sodium mixes, and three amino on MA sulfonic group with AQ on rapidly are combined the formation hydrogen bond, suppressed the redox reaction of AQ, and peak current is reduced.What accompanying drawing 2 showed is that electric current reduces to be worth and the relation between the reaction time.Can find out on scheming, 0.5min after AQ mixes with MA, peak current reduces rapidly, and the speed that reduces of electric current is slack-off gradually afterwards, and after 3min, peak current is substantially constant, shows that AQ is combined with MA fully, and peak current no longer is subjected to the impact in reaction time.Therefore, testing the reaction time of selecting is 3min.
Measured the cyclic voltammogram of AQ in the PBS of different pH.Can find out that from accompanying drawing 3 the redox peak current of AQ is larger as pH=3,4,5 the time, peak shape is better; As pH=6,7,8 the time, the redox peak current of AQ changes little, but that peak shape becomes gradually is irregular.Show that the sensitivity under acid condition of AQ sensor is higher.
Experiment adopts Differential Pulse Voltammetry (DPV) to measure AQ and the peak current of AQ after MA is combined under different pH.When accompanying drawing 4 showed pH=4, not only the peak current of AQ was maximum, and after adding MA, peak current reduces also the most obvious, and when pH=4 was described, the sensitivity of sensor was the highest.This is that when pH=4, MA is positively charged because the melamine isoelectric point is 5.5, and the sulfonic group of AQ is electronegative, both by the electrostatic interaction strong bonded.When system pH increased, melamine presented neutral until electronegative gradually, weakened gradually even with the electrostatic interaction of AQ to disappear, and therefore experiment selects pH=4 to test acidity as the best.
Melamine is with after anthraquinone-2-sodium mixes, and three amino on MA sulfonic group with AQ on rapidly are combined the formation hydrogen bond, suppressed the redox reaction of AQ, and peak current is reduced.Fixedly the concentration of AQ is 1 * 10
-4Mol/L, the concentration that increases gradually MA (increases to 1 * 10 gradually by 0
-4Mol/L) time, the oxidation peak current of AQ is (accompanying drawing 5) that reduces gradually.Relation between the MA that reduces to be worth and to add of oxidation peak current is parabola shaped as shown in Figure 6, complex class Langmuir absorption, and its linear relationship is as shown in Figure 6.
The detection of milk sample
The preparation of positive
Get the milk sample that 25.0 mL do not contain melamine and be placed in the 50mL centrifuge tube, add respectively 5.00 * 10
-9, 5.00 * 10
-8, 5.00 * 10
-7, 5.0 * 10
-6, 5.00 * 10
-5The standard solution of mol/L level, eddy current mixing 2min places 1h, and standard solution is fully absorbed by sample, then divides to install in the 10mL centrifuge tube every pipe 4.0mL, each concentration level packing 6 pipe.
The processing of sample
The trichloroacetic acid solution that adds 300 g/L of 1.2 mL in above-mentioned sample, stir 1 min, with centrifugal 5 min of 3500 r/min, then supernatant is transferred in another centrifuge tube, transfer pH to 4.0 with phosphoric acid solution, standing 1 min, then with the 3500 centrifugal 3min of r/min, take out supernatant standby.
The detection of sample
Adding concentration in the sample is 1 * 10
-4The AQ of mol/L with Differential Pulse Voltammetry oxygen determination galvanic current, draws the MA concentration value of mensuration after three minutes at last according to typical curve.Experimental result (seeing Table 1) illustrates that the method is sensitiveer reliable.
The testing result of table 1 milk sample
Reaction principle of the present invention reduced the oxidation peak current of anthraquinone, and the concentration of the reduction value of electric current and melamine becomes certain linear relationship based on melamine energy and anthraquinone-2-sodium combination.
Accurately pipette the 4.0mL milk sample in the 10mL centrifuge tube, the trichloroacetic acid solution that adds the 300g/L of 1.2mL, eddy current mixes 1 min, with centrifugal 5 min of 3500 r/min, then supernatant is transferred in another centrifuge tube, transfer pH to 4.0 with phosphoric acid solution, standing 1 min, with the 3500 centrifugal 3min of r/min, take out supernatant again, adding concentration is 1 * 10
-4The AQ of mol/L, mixing with Differential Pulse Voltammetry oxygen determination galvanic current, can draw MA concentration value in sample according to typical curve after 3min.
The actual sample measurement result shows, the method is easy and simple to handle, quick, highly sensitive, and whole testing process can be completed in 30min, and the minimum detectability of method can reach 5.00 * 10
-9M (0.6 μ g/mL), the on-the-spot screening in real time that can be applicable to production testing, food inspection, export inspection quarantine detects.
Claims (1)
1. adopt the method for melamine in anthraquinone-2-sodium sensor Fast Measurement sample, comprise following step:
A. prepare the melamine standard solution: take 1.2612 gram melamine standard items, with methyl alcohol dissolving and constant volume in the 10mL volumetric flask, be mixed with the standard solution that concentration is 1 mol/L, be placed under 4 ℃ and keep in Dark Place, be diluted to corresponding concentration with damping fluid again during use;
Preparation anthraquinone-2-sulfonic acid sodium solution: accurately take 0.6205 gram anthraquinone-2-sodium, be settled to 200mL with the phosphate buffer dissolving that pH is 4.0, concentration is 0.1mol/L, being mixed with concentration is the anthraquinone-2-sodium stock solution of 0.01mol/L, is diluted to corresponding concentration with damping fluid again during use;
B. drawing standard curve: add successively a certain amount of melamine standard solution in 6 100mL volumetric flasks, and with 1 * 10
-4Mol/L anthraquinone-2-sulfonic acid sodium solution constant volume makes that in volumetric flask, the concentration of melamine is followed successively by 0 mol/L, 1 * 10
-9Mol/L, 1 * 10
-8Mol/L, 1 * 10
-7Mol/L, 1 * 10
-5Mol/L, 1 * 10
-4Mol/L uses Differential Pulse Voltammetry oxygen determination galvanic current, with the linear relationship drawing standard curve between oxidation peak current reduction value and melamine concentration after three minutes;
C. testing sample preparation:
Solid sample: sample is pulverized, accurately take 5 grams, add 10mL300 g/L trichloroacetic acid solution, eddy current mixing 5min with centrifugal 5 min of 3500 r/min, then transfers to supernatant in another centrifuge tube, transfer pH to 4.0 with phosphoric acid solution, standing 1 min, then with the 3500 centrifugal 3min of r/min, take out supernatant standby;
Fluid sample: the trichloroacetic acid solution that adds 1.2 mL300 g/L in the 25.0 mL testing samples, eddy current mixes 1 min, with centrifugal 5 min of 3500 r/min, then supernatant is transferred in another centrifuge tube, transfer pH to 4.0 with phosphoric acid solution, standing 1 min, then with the 3500 centrifugal 3min of r/min, take out supernatant standby;
D. the detection of sample: adding concentration in the supernatant in the step c is 1 * 10
-4The anthraquinone-2-sulfonic acid sodium solution of mol/L with Differential Pulse Voltammetry oxygen determination galvanic current, draws the melamine concentration value of testing sample to be measured after three minutes at last according to typical curve.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000031101A1 (en) * | 1998-11-23 | 2000-06-02 | Friz Biochem Gmbh | Method for the electrochemical detection of nucleic acid oligomer hybrids |
| US20080044915A1 (en) * | 2004-10-08 | 2008-02-21 | Hunt Donald F | Simultaneous Sequence Analysis of Amino- and Carboxy-Termini |
| CN101413917A (en) * | 2008-10-31 | 2009-04-22 | 扬州大学 | Electrochemical fast detecting method of melamine in dairy produce |
| CN102262115A (en) * | 2011-07-28 | 2011-11-30 | 南京师范大学 | Electrochemical immunosensor for melamine content determination, and preparation method and application thereof |
-
2013
- 2013-01-31 CN CN201310036233.2A patent/CN103115954B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000031101A1 (en) * | 1998-11-23 | 2000-06-02 | Friz Biochem Gmbh | Method for the electrochemical detection of nucleic acid oligomer hybrids |
| US20080044915A1 (en) * | 2004-10-08 | 2008-02-21 | Hunt Donald F | Simultaneous Sequence Analysis of Amino- and Carboxy-Termini |
| CN101413917A (en) * | 2008-10-31 | 2009-04-22 | 扬州大学 | Electrochemical fast detecting method of melamine in dairy produce |
| CN102262115A (en) * | 2011-07-28 | 2011-11-30 | 南京师范大学 | Electrochemical immunosensor for melamine content determination, and preparation method and application thereof |
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