CN109932352A - A kind of Raman detection method of aquatic products Malachite Green and crystal violet - Google Patents
A kind of Raman detection method of aquatic products Malachite Green and crystal violet Download PDFInfo
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- CN109932352A CN109932352A CN201910199058.6A CN201910199058A CN109932352A CN 109932352 A CN109932352 A CN 109932352A CN 201910199058 A CN201910199058 A CN 201910199058A CN 109932352 A CN109932352 A CN 109932352A
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- crystal violet
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Abstract
The invention discloses a kind of Raman detection methods of aquatic products Malachite Green and crystal violet, including sample pre-treatments and Raman detection.Pretreatment process mainly uses liquid extraction method, by extraction, removal of impurities, enrichment, carries out separating-purifying to target molecule in aquatic products.Detection method is by carrying out Raman detection after pre-treatment prepare liquid and gold and silver Nano sol, the interaction of halogen agglomerator, it is identified according to characteristic peak, probe molecular signal under substrate background after can analyze out a variety of different aquatic products pre-treatments, to realize the yin and yang attribute judgement of aquatic products Malachite Green and crystal violet.Detection method proposed by the invention has the characteristics that highly sensitive, high reproducibility, easy to operate, low in cost, to meet the application requirement of malachite green and crystal violet in terms of food science literature.
Description
Technical field
The present invention relates to technical field of analysis and detection, and in particular to a kind of Raman inspection of aquatic products Malachite Green and crystal violet
Survey method.
Background technique
Malachite green (MalachiteGreen oxalate), crystal violet (crystal violet) belong to triphenylmethane
Compound is both dyestuff, and sterilization and parasiticide, to the fungi of fish and fish-egg, bacterium and is posted in aquaculture
It is infested infected with apparent control efficiency.Its Malachite Green can be carcinogenic, and crystal violet is doubtful can be carcinogenic, Non-environmental Pollution Aquiculture neck
Domain country prohibites addition, and malachite green was classified as the banning drugs on aquatic products in 2002 by the Ministry of Agriculture, in aquatic products project not
It must detect.
Due to malachite green and the cheap price and its fish disease prevention and cure effect in aquaculture of crystal violet, cause this two
Person remains incessant after repeated prohibition in aquaculture.2005, Fujian, Jiangxi found malachite green on outlet sea eel, went out to China fish
Mouth produces a very large impact.2010,180 portions of fresh-water fishes detected in Disease Prevention Control Center, Hubei Prov, had respectively
12.22% and 0.56% sample finds the malachite green and crystal violet of different residual degree.
Malachite green and the national standard method of crystal violet are detected in the aquatic products of present to be had: liquid chromatography (GB/
T 20361-2006), Liquid Chromatography/Mass Spectrometry (GB/T 19857-2005), this method sensitivity is higher, but deposit expensive equipment, operation
It is cumbersome, the problems such as pre-treatment step is complicated, pre-treatment higher cost, do not have quick detectability.Anhui Native standard side
Method has used enzyme-linked immunization (DB34/T 1421-2011) and colloidal gold immunity chromatography (DB34/T 2252-2014), the method
It is higher that there are false positives, false positive can not falsfication, the problems such as no detection data saves, and pre-treatment needs to be oxidized aluminium, just oneself
The reagents such as alkane separately purify, grease removal, are an impediment to the purpose quickly detected.
A kind of LR laser raman detection method of malachite green is disclosed in Chinese patent CN103364387B, this method is logical
It crosses after mixing colloidal sol and malachite green and is detected after being dried on aluminium sheet, generally to drying only on micro- focusing Raman
The coffee ring formed afterwards carries out detection just available more stable signal.The method is that a kind of comparison is inefficient, operability
Difference, the uppity detection method of reproducibility.It is far quick, stable without the method detected using colloidal sol.
A kind of rapid detection method of cultivation water Malachite Green, the party are disclosed in Chinese patent CN105675578A
Method detects malachite green using metal-sol and inorganic flocculating agent, but without reference to the aquatic products field of matrix complexity, wherein
Participation of the inorganic flocculating agent to play a crucial role in terms of anti-matrix interference acts on also without reference to explanation, in practical aquatic products product examine
During survey, the road Bu Anqi person not easily passs through the method and obtains Raman signal of the probe molecule under matrix encirclement.
The detection method of a kind of water body and aquatic products Malachite Green, the party are disclosed in Chinese patent CN103105386B
Method is binding molecule engram technology and Magnetic Isolation function, prepares the molecular engram of malachite green on magnetic Nano material surface
Layer is used for trace malachite green separation and concentration, is detected in conjunction with Raman enhancing technology.This method pre-treatment step up to 20
Yu Bu, and it is related to the solid phases such as activation, elution, sample introduction except concentration steps such as miscellaneous operation and rotary evaporations, almost without for quick
The practicability of detection may.
Consider the above problem, the molecular signal performance outstanding in metal-sol in conjunction with malachite green and crystal violet, and
Cleaning action of the first public halogen referred to of this patent in nanoparticle surface to albumen in aquatic products and fat can abandon big
The removal step of part operation complexity, such as aluminium oxide, n-hexane remove fat.And avoid using expensive solid-phase extraction column,
By cheap easy liquid-liquid extraction method, quick detection of the two on aquatic products can be realized.The experiment of one batch multiple groups can be with
Detection is completed in 15 minutes.Under the premise of can distinguish both malachite green and crystal violet signal, for aquatic products mesoporous
Sparrow malachite green detection limit is minimum to reach 5ppb, reaches 2ppb for crystal violet detection limit is minimum in aquatic products.
Summary of the invention
The invention patent aims to overcome that above-mentioned the deficiencies in the prior art, provides a kind of aquatic products Malachite stone
Green and crystal violet Raman rapid detection method, to solve current malachite green and crystal violet in simple, quick, cheap detection side
The problem of face.
Technical solution provided by the invention is as follows:
S1, sample pre-treatments: weighing appropriate homogeneous aquatic products sample, and the water phase acidic extraction solution that 3-10 times of quality is added is super
Sound or extraction of ocean eddies 1-5min are adjusted between PH=5-11 after taking out extracting solution with alkaline reagent, with 0.5-2 times of volume
Low pole or polar organic solvent extraction, take out organic layer after centrifugation layering, then molten with the water phase acid extract of 0.2-1 times of volume
Liquid is stripped organic layer, and water intaking is mutually to be measured after stratification.
Raman detection: appropriate prepare liquid, metal-sol and inorganic agglomerator is added, after mixing in S2 in Raman detection pond
Detection.
Preferably, in the S1, it does not include internal organ, scale and blood etc. that aquatic products sample, which is fish skin and flesh tissue,.
Preferably, in the S1, the solute of water phase acidic extraction solution includes the inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid
With the organic acids such as acetic acid, p-methyl benzenesulfonic acid, solvent includes water or itself and methanol, acetonitrile, ethyl alcohol, acetone, dimethyl sulfoxide, dimethyl
The mixed solution of formamide, hydroxylamine hydrochloride, water phase acidic extraction solution PH=0-4.5 range.The preferred PH=1-2 of inorganic acid, has
The preferred PH=3-4.5 of machine acid.The mixed solution of the preferred water of solvent or itself and acetonitrile, hydroxylamine hydrochloride.
Preferably, in the S1, alkaline reagent include inorganic alkali solution and alkaline buffer solution or its with equivalent solid.It is excellent
Selection of land, PH are adjusted to 6-8.
Preferably, in the S1, low pole or polar organic solvent include: ethyl acetate, petroleum ether, normal heptane, just oneself
Alkane, hexamethylene, t-butyl methyl ether, methylene chloride or its mixed solution.Ethyl acetate, petroleum ether and its mixed solution.
Wherein, in the S1, centrifugation layering requires: 4000-1000rpm is centrifuged 2-5min；It takes out organic phase requirement: avoiding
Get the chip solid on surface layer or middle layer suspension.
Wherein, in the S1, the solute of water phase acid extract solution includes inorganic acid, organic acid, solvent include water or its
With methanol, acetonitrile, ethyl alcohol, acetone mixed solution, water phase acid extract solution PH=1-3 range.
Preferably, in the S2, the metal-sol be nanogold or nano silver, the inorganic agglomerator be halogen in extremely
Few one kind.Preferably, the nano particle diameter of metal-sol is in 50-70nm.
Wherein, in the S2, the concentration of the inorganic agglomerator is 0.01M to its saturated solution.
Preferably, in the S2, the additional proportion of prepare liquid, metal-sol and inorganic agglomerator is 1:0.1-2:0.1-1.
The present invention has the beneficial effect that:
The present invention enhances technology using Raman, using the halogen that simply easily prepares as specific aim agglomerator, by cheap, simple
Just, efficiently pre-treating method realizes the quick detection of malachite green and crystal violet on aquatic products.For aquatic products Malachite
Malachite green detection limit is minimum to reach 5ppb, reaches 2ppb for crystal violet detection limit is minimum in aquatic products, and can pass through characteristic peak area
Divide the two signal.Have into the application that customs port, Shi Yaojiandeng department one line of progress in large quantity, rapidly detect and wants
It asks.
Detailed description of the invention
Fig. 1 is the standard items Raman signal figure of detection method Malachite Green of the present invention and crystal violet；
Fig. 2 is the result figure that malachite green is detected in the embodiment of the present invention 1；
Fig. 3 is the result figure that crystal violet is detected in the embodiment of the present invention 1；
Fig. 4 is the result figure that malachite green is detected in the embodiment of the present invention 2；
Fig. 5 is the result figure that crystal violet is detected in the embodiment of the present invention 2.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
The synthesis of nanogold: the gold chloride of 50mL 0.01% is heated to boiling, and is rapidly added 1% citric acid of 0.5mL
Sodium, high-speed stirred 30min, cooling room temperature.
The synthesis of nano silver: the silver nitrate of 50mL 1mM is heated to boiling, and is rapidly added 1% sodium citrate of 0.5mL,
High-speed stirred 30min, cooling room temperature.
Embodiment 1
The Raman detection of salmon Malachite Green and crystal violet, its step are as follows:
S1 takes the homogeneous sample of 1g salmon, and 5mL HCl solution containing 0.02M, ultrasonic extraction 3min is added；
S2, the supernatant after taking 3mL to extract, is added 0.3mL 0.2M PBS buffer solution (PH=7.4) and 2mL acetic acid second
Ester: petroleum ether (1:1) mix reagent, after vibrating 10s, 5000rpm is centrifuged 3min layering；
S3 takes upper organic phase 1.5mL, and 1mL 10mM HCl back extraction is added, vibrates 10s, stratification；
S4 takes lower layer water phase 0.5mL to be measured；
S5 takes the 100 above-mentioned prepare liquids of μ L in Raman detection pond, 50 μ L nanogold is added, add 50 μ L 0.1M's
NaCl mixes detection, as a result sees Fig. 2, and 3.
Embodiment 2
The Raman detection of fresh water sea eel Malachite Green and crystal violet, its step are as follows:
S1 takes the homogeneous sample of 1g fresh water sea eel, and the 0.1M p-methyl benzenesulfonic acid solution that 5mL contains 20% acetonitrile is added, ultrasound
Extract 3min；
S2, the supernatant after taking 3mL to extract, is added 0.3mL 1M NaOH solution and 2mL methylene chloride: normal heptane (4:1)
Mix reagent, after vibrating 10s, 5000rpm is centrifuged 3min layering；
S3 takes lower layer organic phase 1.5mL, and 1mL is added and is stripped containing 5% ethyl alcohol 1mM HCl, vibrates 10s, stratification；
S4 takes upper strata aqueous phase 0.5mL to be measured；
S5 takes the 100 above-mentioned prepare liquids of μ L in Raman detection pond, 50 μ L nanogold is added, add 50 μ L 0.1M's
KBr mixes detection, as a result sees Fig. 4, and 5.
Above-mentioned is implementation example of the invention, is not able to this and limits design concept of the invention.
The sequencing of above embodiments is not only for ease of description, represent the advantages or disadvantages of the embodiments.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (10)
1. a kind of Raman detection method of aquatic products Malachite Green and crystal violet, concrete operation step:
S1, sample pre-treatments: weighing appropriate homogeneous aquatic products sample, be added 3-10 times of quality water phase acidic extraction solution ultrasound or
Extraction of ocean eddies 1-5min is adjusted between PH=5-11, with the weak pole of 0.5-2 times of volume with alkaline reagent after taking out extracting solution
Property or polar organic solvent extraction, take out organic layer after centrifugation layering, then it is anti-with the water phase acid extract solution of 0.2-1 times of volume
Organic layer is extracted, water intaking is mutually to be measured after stratification；
Raman detection: S2 appropriate prepare liquid, metal-sol and inorganic agglomerator is added in Raman detection pond, detects after mixing.
2. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S1, it does not include internal organ, scale and blood that the aquatic products sample, which is fish skin and flesh tissue,.
3. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S1, the solute of the water phase acidic extraction solution include hydrochloric acid, nitric acid, sulfuric acid, the inorganic acid of phosphoric acid and/or acetic acid,
The organic acid of p-methyl benzenesulfonic acid, solvent include water or/and itself and methanol, acetonitrile, ethyl alcohol, acetone, dimethyl sulfoxide, dimethyl formyl
The mixed solution of amine, hydroxylamine hydrochloride, water phase acidic extraction solution PH=0-4.5 range.
4. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S1, the alkaline reagent include inorganic alkali solution and alkaline buffer solution or its with equivalent solid.
5. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S1, low pole or polar organic solvent include: ethyl acetate, petroleum ether, normal heptane, n-hexane, hexamethylene, tert-butyl
Methyl ether, methylene chloride or its mixed solution.
6. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S1, centrifugation layering requires to be that 4000-1000rpm is centrifuged 2-5min；Take out organic phase and require be avoid getting surface layer or
The chip solid that middle layer suspends.
7. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S1, the solute of water phase acid extract solution includes inorganic acid, organic acid, and solvent includes water or itself and methanol, acetonitrile, second
The mixed solution of alcohol, acetone, water phase acid extract solution PH=1-3 range.
8. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S2, the metal-sol is nanogold or nano silver, and the inorganic agglomerator is at least one of halogen.
9. a kind of Raman detection method of aquatic products Malachite Green and crystal violet according to claim 1, which is characterized in that
In the S2, the concentration of the inorganic agglomerator is 0.01M to its saturated solution.
10. a kind of Raman detection method of aquatic products Malachite Green and crystal violet, feature exist according to claim 1
In in the S2, the additional proportion of prepare liquid, metal-sol and inorganic agglomerator is 1:0.1-2:0.1-1.
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| CN111912929A (en) * | 2020-06-01 | 2020-11-10 | 厦门大学 | Aflatoxin B1Extraction method and application of |
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Application publication date: 20190625 |