CN102608073A - Quick detection method for melamine in dairy products - Google Patents

Abstract

The invention relates to a quick detection method for melamine in dairy products. Based on the principle of three intermolecular hydrogen bonds between melamine and thymine, the method is set up for quickly and flexibly detecting melamine in dairy products under an aqueous solution buffer system by the aid of gold nano particles wound by polyT55. The specific hydrogen bonds between the melamine and the polyT55 enable the gold nano particles wound by the polyT55 to be aggregated so as to cause variation of PRLS (prolactin signal) of gold size, and detection of melamine in dairy products is realized. The method is ingenious in design, high in selectivity and flexibility, free of modification, simple in operation and free of large instruments.

Description

The method for quick of melamine in a kind of dairy produce

Technical field

The present invention relates to the melamine detection method.

Background technology

Melamine is entrained in milk or the animal feed to reach the purpose of increasing percent protein because itself nitrogen content up to 66%, is often utilized by illegal merchant.The melamine of this illegal interpolation in milk or animal feed has caused baby's kidney stone or even death incident, thereby people pay close attention to melamine detection very much recently.

The method that detects melamine at present mainly contains high-efficient liquid phase technique (HPLC), high performance liquid chromatogram and mass spectrometry methods such as (HPLC-MS), yet these methods relate to large-scale instrument, is unfavorable for that real-time analysis fast detects.A kind of new method simple to operate, sensitive, the fast detecting melamine of present situation development to present conventional detection is highly significant.

Summary of the invention

The object of the invention is exactly the deficiency that exists to prior art, based on the principle of melamine and intermolecular three hydrogen bonds of thymine (T base), under the buffer system of the WS, utilizes polyT ₅₅The gold nano grain that twines has been set up a kind of method that detects melamine in the dairy produce fast, delicately, and this method has than high selectivity and sensitivity, is excused from a college course decorations, and is simple to operate, do not need large-scale instrument; Simultaneously, this method has also been expanded three application of hydrogen bond structure in the system of water analyzing and testing of melamine-thymine pattern.

Usually three hydrogen bond structures of melamine-thymine pattern are only at solid phase surface, meteorological liquid interface, in the organic phase or the ultravacuum condition could form.The applicant has designed under aqueous conditions, sets up a kind of method that simply detects melamine fast based on the principle of melamine and intermolecular three hydrogen bonds of thymine.

At present, this three structure of hydrogen bonds be applied in the analyzing and testing system of aqueous phase report seldom.The applicant finds; In the base of four kinds of DNA; Have only the T base to form this similar three structure of hydrogen bonds, so the applicant is material with the gold nano grain, invented this method with melamine in very high specificity, the highly sensitive and visual detection dairy produce with melamine.

The inventive method step is following:

1. operation steps

(1.1) handle milk sample: use dissolved in purified water to being 50 mL totally earlier milk sample, use the ultra filtration membrane ultrafiltration after diluting 10 times more afterwards, behind centrifugal 30 min of 20000 rpm, get the upper strata stillness of night and dilute 10000 times;

(1.2) DNA polyT ₅₅With hatching of gold nano grain AuNPs: in centrifuge tube, adding mol ratio is gold nano grain AuNPs and the DNA polyT of 1:23 _55,, the whirlpool mixes, room temperature reaction 5-10 min;

(1.3) and then successively adding needs dairy produce that detects and pH 7.6 NaH that are equivalent to solution mixture cumulative volume 1/5 ₂PO ₄-Na ₂HPO ₄Buffer solution, vortex mix back 5-10 min;

(1.4) adopt PRLS to measure, when measuring scattering spectrum, excitation wavelength and emission wavelength are set equate, promptly

_Em=

_Ex

2. interpretation of result:

(2.1) spectral signature: if at 520 nm places the plasma resonance absorption peak is arranged, the scattering strength at 550 nm places strengthens gradually, and the absorption at the 620nm place also presents the linear trend that strengthens, and then shows and contains melamine in the dairy produce;

(2.2) metamorphosis:, or become redness to carry out the transition to purple to become blueness at last, also show and contain melamine in the dairy produce if observe gold nano grain solution and become redness.

Principle of the present invention is shown in Figure 1 as illustrating, polyT ₅₅Be wrapped in gold nano grain AuNPs surface, gold nano grain is in disperse state; After adding melamine, because the effect of melamine and intermolecular three hydrogen bonds of thymine, gold nano grain is assembled.Utilize the gathering disperse state of gold nano grain to reach the purpose that detects melamine.

For assessing the selectivity that this method is used for practical measurement, investigate to the interference of inorganic ions common in the milk and some concurrents, have only protein that this detection is had certain interference.Because albumen can be adsorbed on the gold nano grain surface and shield and make the gold nano grain aggregation extent die down.Thereby we are when milk sample detects, and go out the bigger albumen of molecular weight with the mode of ultrafiltration earlier and prevent that its gathering to gold nano grain from exerting an influence.

Under optimum experimental condition, tie up to the PRLS intensity under the different melamine concentration according to this method measuring body, and with 560.0 nm places

I _PRLSTo the melamine concentration mapping, equation of linear regression is:

I _PRLS=-11.2+141.0 c(melamine, nM), related coefficient is 0.9905, and the range of linearity is 80 nM-1000 nM, and the detection of this method is limited to 20 nM.Under the similarity condition, the ratio and the melamine concentration that absorb according to 620 nm places and 520 nm places also can draw certain linear, but the range of linearity of detection this moment is 150 nM-800 nM.It is thus clear that under this experiment condition, the remolding sensitivity PRA signal of PRLS signal highly sensitive, it is lower that it detects melamine concentration.

The advantage and the good effect of invention:

The present invention is based on the principle of intermolecular three hydrogen bonds of melamine and thymine, under the buffer system of the WS, utilize polyT ₅₅The gold nano grain that twines has been set up a kind of method that detects melamine in the dairy produce rapidly and sensitively.Wherein, melamine and polyT ₅₅Between specific hydrogen bond action make and to twine polyT ₅₅Gold nano grain assemble, thereby the PRLS signal that has caused gold size changes, thereby realize melamine detection in the dairy produce.

This method design is ingenious, has than high selectivity and sensitivity, is excused from a college course decorations, and is simple to operate, do not need large-scale instrument; From another angle, we have expanded the application of three hydrogen bond structures in the analyzing and testing system of the WS of melamine-thymine pattern, can predict that the pattern of this three hydrogen bonds will be more widely used in analysis system.

Description of drawings

Fig. 1: the contrast synoptic diagram of invention scheme and HPLC testing result;

Annotate: poly-T _n.poly-A _n, ploy-C _n, poly-G _nRepresent that respectively dna molecular is with gathering the thymine oligonucleotides, with gathering the adenine oligonucleotides, with gathering the cytimidine oligonucleotides, with gathering the guanine oligonucleotides.Gold nano grain is a gold size, and Melamine is a melamine, and Thymine is the T base.

Fig. 2: plasma resonance absorbs the result.

Fig. 3: plasma resonance scattering result.

Embodiment

1: handle milk sample (milk sample I; Milk sample II; Milk sample III), it totally is 50 mL that 2 mg milk powder or 2 mL liquid milks use secondary water to be dissolved to earlier, dilutes after 10 times the ultra filtration membrane ultrafiltration with 10 KD afterwards again; Behind centrifugal 30 min of 20000 rpm, get 10000 times of upper strata stillness of night dilutions.

2: get 30 μ L 1.0 * 10 ^-5M melamine solution (37.8 ng) joins respectively in the above-mentioned ultrafiltration diluted milk of the 1.0 mL appearance.

In the centrifuge tube of 1.5 mL, adding mol ratio is gold nano grain AuNPs and the DNA polyT of 1:23 ₅₅(the final concentration 2nM of gold nano grain AuNPs, DNA polyT ₅₅Final concentration 0.046uM), the whirlpool mixes, room temperature reaction 5-10 min.Join in the mixed solution of milk and melamine again, detect with the PRLS method, excitation wavelength and emission wavelength equate when measuring scattering spectrum, promptly

_Em= _Ex

2, analysis result:

(1) through the spectrogram analysis: as shown in Figure 2, melamine ability and poly-T ₅₅T base specific ground in the dna molecular forms three hydrogen bonds, causes gold nano grain to be assembled, and is attended by change in color, and obvious variation has taken place for spectral signature and form.At first, polyT ₅₅Be wrapped in the gold nano grain surface; Make it be in disperse state, solution colour takes on a red color, and the plasma resonance absorption peak of its characteristic is arranged at 520 nm places; After the melamine of variable concentrations adds this system; Gold nano grain solution carries out the transition to purple from redness and has become blueness at last, and the absorption at the 620nm place also presents the linear trend that strengthens, and this is the variation that gold size is assembled the typical absorption peak in back.Simultaneously, gold nano grain strengthens in the scattering strength at 550 nm places gradually, and the enhancing value also presents the certain linear (see figure 3) with the amount of the melamine that adds.

(2) pass through color analysis:

Contrast with the HPLC method, the result is following: determine content of melamine in the HPLC method and be respectively 37.5 ng/mL, 39.5 ng/mL, 39.9 ng/mL; And PRLS method mensuration result is 41.0 ng/mL, 37.4 ng/mL, and 39.0 ng/mL, more identical.

We have determined the content of melamine in three kinds of milk samples in this way, measure the HPLC method contrast of result and National Standard Method, the mensuration reliable results.

 

The contrast of melamine and HPLC method testing result in table 1 plasma resonance scattering method (PRLS) the detection milk

。

Claims (1)

1. the method for quick of melamine in the dairy produce, said mode is under the buffer system of the WS, utilizes polyT ₅₅The gold nano grain that twines is assembled disperse state and is reached the purpose that detects melamine, and the step of said method is following:

(1) operation steps

(1.1) handle milk sample: use dissolved in purified water to being 50 mL totally earlier milk sample, use the ultra filtration membrane ultrafiltration after diluting 10 times more afterwards, behind centrifugal 30 min of 20000 rpm, get supernatant liquor and dilute 10000 times;

(1.2) DNA polyT ₅₅With hatching of gold nano grain AuNPs: in centrifuge tube, adding mol ratio is gold nano grain AuNPs and the DNA polyT of 1:23 _55,, the whirlpool mixes, room temperature reaction 5-10 min;

(1.3) and then successively adding needs dairy produce that detects and pH 7.6 NaH that are equivalent to solution mixture cumulative volume 1/5 ₂PO ₄-Na ₂HPO ₄Buffer solution, vortex mix back 5-10 min;

(1.4) adopt PRLS to measure, when measuring scattering spectrum, excitation wavelength and emission wavelength are set equate, promptly

_Em=

_Ex

(2) interpretation of result:

(2.1) spectral signature: if at 520 nm places the plasma resonance absorption peak is arranged, the scattering strength at 550 nm places strengthens gradually, and the absorption at the 620nm place also presents the linear trend that strengthens, and then shows and contains melamine in the dairy produce;

(2.2) metamorphosis:, or become redness to carry out the transition to purple to become blueness at last, also show and contain melamine in the dairy produce if observe gold nano grain solution and become redness.

Images