CN103512789B - A kind ofly extract the reagent of analyte and the method for extraction in sample - Google Patents
A kind ofly extract the reagent of analyte and the method for extraction in sample Download PDFInfo
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- CN103512789B CN103512789B CN201210214022.9A CN201210214022A CN103512789B CN 103512789 B CN103512789 B CN 103512789B CN 201210214022 A CN201210214022 A CN 201210214022A CN 103512789 B CN103512789 B CN 103512789B
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- extraction
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- analyte
- agent
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Abstract
The invention provides a kind of reagent processing sample before detection, this reagent comprises: extraction auxiliary agent, allowed by this extraction auxiliary agent and be in analyte in sample and this sample separation,? or/and allow analyte be non-band electrical property, or/and in the environment allowing analyte be in be ostracised; Extraction agent, is extracted analyte by extraction agent from the environment after being extracted auxiliary agent process; Nonpolar correctives, by adding nonpolar correctives by changing the solubleness of analyte in the extract be separated, allows analyte be assigned in immune response compatibilizing agent, for follow-up immunology detection.
Description
Technical field
The present invention relates to a kind of reagent and the disposal route that process sample before detection, especially, the present invention relates to a kind of reagent and disposal route detecting analyte pre-treatment sample in sample with immunization method.
Background technology
First immunology detection technology grows up from clinical examination field, and it mainly detects sample is blood, urine, the liquid type samples such as saliva.These liquid type samples general directly can be used to immunology detection.And in field of detection of food safety, generally needing analyte, such as some small-molecule substances, from tissue samples, such as liver, fat, kidney etc., middlely first extract, and is used further to follow-up detection.
In analyte extraction, also there is the method that some are traditional, such as, soxhlet extraction, it is applicable to the project of muting sensitivity detection means, need the tested substance in sample to be extracted as far as possible, also have requirement, to meet as conventional methods such as traditional spectroscopic assaies to the purity after it extracts.Such as liquid-liquid extraction method again, the method extracts between low pole and intensive polar solvent, but needs a large amount of organic solvents could meet the requirement detected.
The confirmation method of the agricultural and veterinary chemicals residue detection of generally acknowledging in the world is at present instrumental method, comprises liquid chromatography, gas chromatography, gas chromatography mass spectrometry and LC-MS.This respect, in order to adapt to the requirement of instrument, normal use C18 or HLB(hydrophilelipophile multipolymer) solid-phase extraction column purify sample, also has molecular engram polymeric material (MIP) in addition as enrichment material (superpolymer in three holes of the function base be combined with target molecule selectivity), it is in fact a kind of bionics principle, and namely Artificial Antibodies identifies and catches the epi-position with spatial complementary.In recent years, some large biotech firms, utilize antibody technique to prepare immune affinity chromatographic, for the sample clean before the detection of instrumental method.
Compared with these this traditional method, at present, the extracting method of the analyte in tissue samples, in extraction efficiency, uses in quantity of solvent and has larger improvement, but there is the problem of inefficiency.Have scholar also exploring the method for raising the efficiency, as high flux, 96 hole extraction solid-phase extraction devices, within one day, can only process 24 samples, efficiency is lower.
Carrying out about using instrument analyzing the pretreatment technology used, also constantly making progress at present, as supercritical extract, gel permeation chromatography, accelerated solvent extraction, solid-phase microextraction, matrix solid phase micro-extraction etc.
In a word, detect for traditional instrument, the pre-treating method of the analyte in complicated tissue samples is a test greatly, and even, some pre-treating method is only applicable to a kind of detection of tissue, and another kind is organized, and can not be suitable for.This chief reason is the detection material means of instrument itself is chromatogram and mass spectral characteristic, and the signal to noise ratio (S/N ratio) of these features is in low-down concentration (10
-9mol/L) under be difficult to stable maintaining.
At present, although there are some immunoreagents for food safety detection, such as ELISA kit, not only colloid gold test paper etc., have solved efficiency and the accuracy problem of detection to a certain extent, still can not accomplish efficient but also effective in pre-treatment.About how to extract Small molecular in the sample of complexity, current immunological method also Primary Reference is tending towards ripe instrumental method pre-treating method, and even many methods are direct reference instrument methods.
For chloromycetin, the Ministry of Agriculture 1025 announces residual chloromycetin in-26-2008 animal-derived food and detects the muscle of enzyme linked immunosorbent assay, the pre-treating method of liver and fishes and shrimps sample, for taking test portion 3g, adds ethyl acetate (organic solvent) 6ml and vibrates, centrifugal, get upper organic phase, nitrogen dries up, residue n-hexane dissolution, then adds buffer solution intense oscillations, centrifugal, take off layer (non-normal hexane) layer liquid and carry out immunoassay.The method and the Ministry of Agriculture announce the mensuration gas chromatography-mass spectrography of chloramphenicol residue in 781-1-2006 animal-derived food, the mensuration high performance liquid chromatography-tandem mass method of chloramphenicol residue in No. 781, Ministry of Agriculture bulletin-2-2006 animal-derived food, in No. 1025, Ministry of Agriculture bulletin-21-2008 animal-derived food, the extracting method of residual chloromycetin detection vapor-phase chromatography is basically identical, its core remains and uses ethyl acetate to be separated with impurity by target molecule chloromycetin, under the prerequisite ensureing effectively extraction, nitrogen is utilized to dry up removal ethyl acetate, recycling diverse ways gets rid of the interference to subsequent detection, thus reach certain accuracy.
For beta-2-agonists class Ractopamine, in No. 1025, Ministry of Agriculture bulletin-6-2008 animal food, Rct opamine residue detects enzyme linked immunosorbent assay employing acetonitrile and does the Ractopamine Small molecular extracted in tissue, dry up through nitrogen, step of redissolving obtains the sample that can be used for euzymelinked immunosorbent assay (ELISA) mensuration, the extracting method flow process announced in-18-2008 animal derived food in beta-receptor activator residue detection Liquid Chromatography-Tandem Mass Spectrometry with No. 1025, the Ministry of Agriculture is substantially similar, i.e. organic solvent extraction, remove organic solvent, again residue is dissolved in examination with computer in the solvent being suitable for instrument detection.
For Nitrofuran metatolites, in DB33T744-2009 aquatic products furazolidone, AMOZ Fast Measurement euzymelinked immunosorbent assay (ELISA) in first derivatization is carried out to sample, re-use ethyl acetate furan derivative is extracted, remove ethyl acetate, this place uses nitrogen to dry up, target substance is dissolved out, for immunoassays by recycling normal hexane and biocompatibility buffer solution.This kind of method and No. 1077, the Ministry of Agriculture announce the mensuration high performance liquid chromatography of Nitrofuran metatolites residual quantity in-2-2008 aquatic products, in No. 783, Ministry of Agriculture bulletin-1-2006 aquatic products, the extracting method of the mensuration Liquid Chromatography-Tandem Mass Spectrometry of Nitrofuran metatolites residual quantity substantially all follows extraction and the method such as nitrogen or Rotary Evaporators removal ethyl acetate, then carries out next step determination step.
Adopt immunology detection, although can realize without equipment at follow-up detection-phase, high-throughout detection, just the whole method of inspection, does not significantly improve the efficiency of inspection.The main cause that this problem produces is, step at present for the pre-treating method of immunology detection mainly still considers the detection adapting to instrument, block chromatographic column as prevented and take suitable solvent to remove protide in sample or lipid material, in order to the instruments and methods analysis making the material to be detected detected in sample easily be given physicochemical property, usually adopt purification, and derivatization step.In addition, these Small molecular are not be free in sample tissue but be organically combined by various chemistry or physical action with these sample tissue, Small molecular and these sample tissue is allowed effectively to dissociate out, and not affecting follow-up immunology detection, current method can only be realized by the method drying up organic solvent.
Recently, this immunoassay device is adopted by average family or amateur mechanism more and more.Because these check and evaluation aim at designed by layman, so these pick-up units need operating process simple, and the accuracy of testing result can be ensured.Therefore, simple to operate and testing result accurately and reliably pick-up unit be present society institute in the urgent need to.
Summary of the invention
In order to solve these defects that traditional product exists, the invention provides a kind of before detection to the reagent of tissue samples process and the method adopting agent treated sample, can simplify processes process greatly by the method, operate for amateur occurrences in human life.Especially, in time adopting immunoreagent to detect analyte, efficiency and the accuracy of detection can be improved.
On the one hand, the invention provides a kind of reagent processing sample before detection, this reagent comprises: extraction auxiliary agent, is allowed being in analyte in sample and this sample separation by extraction auxiliary agent, or/and allow analyte be non-band electrical property, or/and analyte is in the environment of being ostracised; Extraction agent, allows analyte be extracted in extraction agent from the environment after being extracted auxiliary agent process by this extraction agent.
In some preferred modes, preferably, extracting the use of auxiliary agent occurs in after sample contacts with extraction agent.Certainly, extraction auxiliary agent and both extraction agent also can contact with sample tissue simultaneously and mix, or carry out homogenized after mixing.
In some preferred modes, extraction agent is organic extraction reagent.In some preferred modes, the similarity degree of the analyte under the polarity of extraction agent and non-electriferous state is higher than sample and extract auxiliary agent.Preferred, this extraction agent or partial extraction reagent and sample and extract auxiliary agent can not be miscible.In the preferred mode of other, this extraction agent possesses low toxicity or nontoxic, chemical inertness, and without the quick-fried toxicity matter processed of system.These extraction agent can be ester class, nitrile, and the potpourri of ester class and alcohols or ethers.
Reagent of the present invention can further include nonpolar adjustment reagent, and this nonpolar adjustment reagent and extraction agent can miscible formation miscible liquids.Preferably, the similarity degree of the target analyte under the polarity of this nonpolar adjustment reagent and non-electriferous state is lower than extraction agent.In some preferred modes, nonpolar adjustment reagent comprises hydrocarbon compound.Preferred, nonpolar adjustment reagent comprises fat hydrocarbon and/or the corresponding derivant of fat hydrocarbon.These nonpolar adjustment reagent include, but are not limited to, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, tridecane, dodecane, sherwood oil, decahydronaphthalene, terebinthina, gasoline, kerosene etc.
In the preferred mode of other, reagent of the present invention can further include some immune response intermiscibility reagent.Preferably, the miscible liquids of immune response compatible solutions and non-polar solvent and the miscible formation of extraction agent is immiscible.
Another aspect, the present invention also provides a kind of kit processing sample, and this kit comprises: extraction auxiliary agent, is allowed being in analyte in sample and this sample separation by extraction auxiliary agent, or/and allow analyte be non-band electrical property, or/and analyte is in the environment of being ostracised; Extraction agent, allows analyte be extracted in extraction agent from the environment after being extracted auxiliary agent process by this extraction agent; Nonpolar adjustment reagent, this nonpolar adjustment reagent and extraction agent can miscible formation miscible liquids; With immune response intermiscibility reagent.
In some preferred modes, extraction auxiliary agent, extraction agent, nonpolar adjustment reagent and immune response intermiscibility reagent exist separately respectively and mutually do not mix, and such as above four kinds of reagent are contained in container separately.In other mode, extraction auxiliary agent and extraction agent are contained in a container, and such as glass container or plastic containers, nonpolar adjustment reagent and immune response intermiscibility reagent are carried in another container by splendid attire, such as glass container or plastic containers.
On the other hand, the invention provides a kind of method processing sample before detection, the method comprises the following steps:
(1), allow extraction auxiliary agent and sample contact, this extraction auxiliary agent is by the analyte in sample and sample separation; And/or allow analyte be non-band electrical property, or/and in the environment allowing analyte be in be ostracised;
(2), allow and to be contacted with extraction agent by sample, allow analyte be extracted in extraction agent;
(3), allow the extract be separated contact with immune compatibility reagent with nonpolar correctives, allow analyte be assigned in immune compatibility reagent.
In some preferred modes, can first contact with extraction auxiliary agent in the sample, and then and extraction agent; Contact with extraction auxiliary agent; Or, allow sample first contact with extraction agent, and then contact with extraction auxiliary agent; Or extraction auxiliary agent and extraction agent can simultaneously and sample contact.
In some preferred modes, allow the similarity degree of the analyte under the polarity of extraction agent and non-electriferous state higher than sample and extract auxiliary agent.Preferred, allow extraction agent or partial extraction reagent and sample and extract auxiliary agent and can not mix.Or allow extraction agent or partial extraction reagent and sample and extract auxiliary agent can layering.
In other preferred modes, the method is also included in extraction agent and adds nonpolar adjustment reagent, allows nonpolar adjustment reagent and extraction agent can miscible formation miscible liquids.Preferably, allow the similarity degree of the interesting small organic molecules under the polarity of nonpolar adjustment reagent and non-electriferous state lower than extraction agent.
In the preferred mode of other, method of the present invention can further include in the miscible liquids formed at nonpolar adjustment reagent and extraction agent and adds some immune response compatibilizing agents.Preferably, the liquid allowing immune response compatible solutions and non-polar solvent and extraction agent be mixed to form is immiscible.
At immune response compatible solutions, extraction agent, and in the mixed process of non-polar solvent, analyte, will be ionized, the effect of hydrogen bond etc., redistribute back in immune response compatible solutions, finally realize the detection to analyte.
In above aforesaid all embodiments, extraction auxiliary agent can comprise some inorganic salts.These inorganic salts comprise, but are not limited to, ammonium sulfate, sodium sulphate, magnesium sulfate etc.Preferred, these inorganic salts are high concentration, and such as inorganic salts is the inorganic salts being greater than 20% the solubleness of 10-50 DEG C with water, comprise sodium, potassium, caesium, magnesium, calcium, copper, silver, iron, the hydrochloric acid of ammonium, nitric acid, sulfate, and the alkaloids that to meet dissolubility be water is greater than 20% the solubleness of 10-50 DEG C.
In above aforesaid all embodiments, sample can be 0.2-4 with the volume ratio of extraction auxiliary agent.
In above aforesaid all embodiments, the potpourri of sample/extraction auxiliary agent and the volume ratio of extraction agent can be 0.2-10.
In above aforesaid all embodiments, the extract of separation and the volume ratio of nonpolar correctives are 0.2-10.
In above aforesaid all embodiments, sample can be the tissue of animal body, such as musculature, adipose tissue, visceral organ tissue, hair tissue; Or comprise blood, blood plasma, serum, sweat, saliva, the body fluid such as urine.
In above aforesaid all embodiments, analyte can comprise any small-molecule substance as medicine, such as some small molecule organic compounds.Small molecule organic compound can be hormone medicine, microbiotic, and industrial chemical.Small molecule organic compound can be, but be not limited to, beta-2-agonists class medicine, chloromycetin, sulfa drugs, tetracycline, nitrofurans metabolin, fluoroquinolones, beta-lactam antibiotic, malachite green, concealed malachite green, crystal violet, melamine, olaquindox, 19-nortestosterone, Trenbolone, norethindrone, diethylstilbestrol, Florfenicol, medroxyprogesterone acetate, estradiol, or one or more in stabilizing.
In above aforesaid all embodiments, immune response compatibilizing agent or solution include, but are not limited to, phosphate buffered solution, citric acid solution, carbonate buffer solution, MES, TRIS, HEPES, MOPES, physiological saline.
In above aforesaid all embodiments, detect the detection comprising any means, such as, comprise liquid chromatography, gas chromatography, the instrumental method such as gas chromatography mass spectrometry and LC-MS.Detection method also comprises the immunology detection based on immunology principle, such as, based on enzyme-linked immunoassay method and the cross flow reagent strip method of antibody and antigen combination principle, and with these immunization methods with the use of instrument read method.
Beneficial effect
Utilize reagent of the present invention effectively can extract analyte from sample, make follow-up detection consistent with real level, improve the accuracy of detection; Because which reduce traditional concentrated and evaporation, utilize reagent of the present invention and disposal route not to need complicated step and complicated utility appliance, the people generally without professional standards can operate; In addition, the analyte using reagent of the present invention and method to extract can directly utilize immunological detection method to detect, and can well coordinate, be beneficial to popularization with some current commercially available immunologic detection methods.
Of the present inventionly to elaborate
The invention provides the using method of a group reagent and this group reagent.By the use of this group reagent, can by 10
-5below mol/kg sample, especially, 10
-7mol/kg sample 10
-10the small molecule organic compound of mol/kg sample, mainly comprises various agricultural chemicals, veterinary drug, hormone, and environmental contaminants are residual to be extracted, for immunology detection from biological specimen.
So-called sample, mainly comprises animal tissue, dairy products, birds, beasts and eggs, and various body fluid, and hair.
So-called extraction agent refers to middle polarity, chemical inertness, insoluble or be slightly soluble in water, or can not with the miscible solvent of arbitrary volume ratio with water.
So-called nonpolar adjustment reagent, itself and extraction agent have low pole or the non-polar solvent of good compatibility.
So-called extraction auxiliary agent, refers to energy and water immiscible liquid, includes but not limited to strong acid, example hydrochloric acid; Or highly basic, as NaOH; Or oxygenant, as sodium nitrite, or protein denaturant, as urea; Or acid or alkaline buffer solution, as glycine buffer, Tris damping fluid; Or Lewis acid, as aluminium choride; Or Lewis alkali, as ammoniacal liquor; Proteinase, as pepsin; Or the inorganic salts that under normal temperature and pressure environment, solubleness is high in water; And one or more mixed solutions as above.
So-called layering, refer to that out of phase outward appearance is distinguished obviously, method comprises standing, centrifugal, but is not limited to leave standstill, or centrifugal method.
Analyte can be organic molecule medicine, so-called organic molecule medicine: molecular weight is lower than 1000 daltonian organic compounds, and it has a certain impact to the physiological activity tool of living organism.
So-called residual: in edible animal derived food, residual medicine, or the metabolin of medicine, content is lower than below 10-7 mol/kg of sample.
So-called immune response intermiscibility solution refers to conventional immunological response buffer solution, and with the potpourri of particular matter, this solution not appreciable impact immunological response and follow-up detection.
Characteristics of principle one of the present invention is: extraction auxiliary agent, and its function includes but not limited at strong acid, highly basic, oxidation/reduction, or dissociates out by the covalent bond small molecule organic compound in tissue to be checked under the effect of enzyme; Include but not limited under the effect of denaturant, by the structural failure of physisorption Medicine small molecule in tissue to be checked; Include but not limited to change the band point patterns of Small molecular at self, make it be tending towards being assigned in suitable extraction agent.
Characteristics of principle two of the present invention is: suitable extraction agent, with sample and/or extract auxiliary agent and fully mix, utilizes external force, includes but not limited to centrifugal, or stratification, interesting small organic molecules is extracted to extraction agent mutually in.
Characteristics of principle three of the present invention is: the nonpolar correctives of certain proper proportion and extraction agent and immune response compatible solutions fully mix, utilize external force, include but not limited to centrifugal, or leave standstill make interesting small organic molecules be dissolved in immune response compatible solutions, for follow-up immunological test.
analyte
Analyte can be organic molecule, as medicine organic molecule comprise agricultural chemicals, veterinary drug, hormone and environmental contaminants etc., and the compound of some carbon elements (beyond the oxide, carbonic acid, carbonate, cyanogen, prussiate, oxygen cyanogen, cyanate, sulphur cyanogen, metal carbide etc. of de-carbon) is referred to as organism.It is generally acknowledged that molecular weight is lower than 1000 daltonian molecules, is Small molecular.Mainly to refer in breeding process, for promoting growth of animal or treatment Animal diseases and the medicine that artificially adds, be mainly microbiotic, sulfa drugs, nitrofurans, anthelminthic chemotherapeutic product, hormone etc. for veterinary drug.
As the drug effect essence of the organic molecule of medicine
Observe from macroscopic scale, to interact the reaction produced with body, be i.e. medicament contact or after entering body, promote that the biochemical functions of body surface and internal environment changes, or suppress the pathogen of invasion, assist body to improve resistance against diseases, reach the effect of disease preventing and treating.As the explanation of microscopic scale, after the ad hoc structure being known as medicine and body or pathogen at present be combined with each other and acts on, body plays corresponding curative effect in conjunction with self-regulation function.The core of the combination of medicine and body or pathogen is effective combination of drug molecule and corresponding target position.Acting force between drug molecule and body or pathogen target position has two aspects to originate, and be the complementarity of space three-dimensional structure on the one hand, derives from the interaction in the corresponding site of complementary structure on the other hand, and combine the stability of ligand afterwards.
The essence that organic molecule and body tissue sample (can be called part again) combine.
Combination between Small molecular and tissue samples is that the biomacromolecule surface of body or pathogen exists the target position structure with medicine complementation, and often the essence of this structure is protein naturally folding of amino acid chain and the result of the specific conformation produced in physiological conditions of body or pathogen self, simultaneously, in conjunction with lipid and glucide on the amino acid residue of protein, also can form specific conformation in physiological conditions, be combined with medicine generation non-covalent under certain drug concentration.Utilize these principles, under the small-molecule substance with part can be allowed to be in extreme condition, as strong acid, highly basic, high salt, under the effect of scaling agent, by destroying the conformation of the part be combined with medicine, makes Small molecular dissociate out from sample.
The essence that small molecule organic compound dissolves
Depend mainly on the character of small-molecule substance itself, in extreme circumstances, the protein of tissue will be entirely destroyed, thus lose the character with Small molecular specific binding, in different phase-splitting, micromolecular distribution, depends on Small molecular distribution character between different phases itself.According to the similar principle that mixes, Small molecular in the not abundant mixed distribution of homophase, Small molecular be tending towards polarity similar mutually in.This in the following description will be detailed elaboration.
When the concentration 10 of analyte
-7mol/kg sample 10
-10during mol/kg sample, organic molecule medicament residue in the tissue due to the target position Non-covalent binding under normal physiological condition, or metabolin and the covalent bond of reactive group under the catalysis of biology enzyme, present 10
-7mol/kg sample-10
-10mol/kg concentration of specimens and high dispersion, generally can not exist with crystalline state.Therefore micromolecular extraction essence is to select suitable extraction agent in this case, and destroys and the body of its complementation or pathogen part.
When low concentration organic molecule, as 10
-7mol/kg sample-10
-10mol/kg or after following small-molecule substance is extracted, by adding a certain amount of nonpolar adjustment reagent, with immune compatibility solution, the solvation of extraction agent to organic molecule can be changed, under suitable formula and ratio situation, the organic molecule of low concentration will be redistributed back in immune response compatible solution, at this moment may be used for the follow-up high sensitivity testing method based on immunological response.
sample
Any tissue that may contain analyte can as sample, body tissue such as containing organic molecule medicine also can as sample, these body tissues can be the tissues of animal body, such as musculature, adipose tissue, visceral organ tissue, hair tissue; Also can be comprise blood, blood plasma, serum, sweat, saliva, the body fluid sample of urine etc.Animal tissue can be any animal, such as, as various cultivated animals or the wild animal of food, and such as pig, sheep, chicken, duck, the animals such as ox.
extraction auxiliary agent
In the present invention, at sample to be measured, after such as, adding extraction auxiliary agent in tissue samples, carry out physical mechanical vibration.Extract auxiliary agent in this process and will produce irreversible destruction to organic molecule plementary ligand structure in sample.Owing to extracting the strong acid of the high concentration (usual final concentration is at about 0.5M) of auxiliary agent, example hydrochloric acid, or highly basic, as NaOH, or oxygenant, as sodium nitrite, the protein born with the effect of Small molecular plementary ligand structure stand will be hydrolyzed in sample, protein chain is ruptured.Analyte is allowed to be non-band electrical property, or/and in the environment that allows analyte be in be ostracised.Or protein denaturant, as urea, under existence, make protein further sex change occur.In the extreme case, the ligand structure be originally combined with Small molecular there occurs irreversible change, and the product after change will pass through various weak force at this kind of extreme environment again, as Van der Waals force, hydrogen bond, sat linkage, electrostatic interaction and water delivery effect, be combined with each other again.Thisly to be combined with each other, do not possess physiological characteristic, on the one hand due to the flexibility of chain structure, make this interaction very strong, eliminate the possibility that small-molecule substance combines with it on the other hand.Simultaneously under this kind of environment, organic molecule presents non-band electrical property, and the electrostatic interaction of the material in Small molecular self and environment is further weakened.Such Small molecular thoroughly separates with the part of its complementation, thus dissociates out.
Meanwhile, extraction auxiliary agent comprises the inorganic salts containing high concentration, as ammonium sulfate.Extraction auxiliary agent can comprise some inorganic salts.These inorganic salts comprise, but are not limited to, ammonium sulfate, sodium sulphate, magnesium sulfate etc.Preferred, these inorganic salts are high concentration, and such as inorganic salts is the inorganic salts being greater than 20% the solubleness of 10-50 DEG C with water, comprise sodium, potassium, caesium, magnesium, calcium, copper, silver, iron, the hydrochloric acid of ammonium, nitric acid, sulfate, and the alkaloids that to meet dissolubility be water is greater than 20% the solubleness of 10-50 DEG C.Inorganic salts contribute to being separated of sample and extraction agent, and regulate the extraction efficiency of organic molecule.
In addition, in the environment of extraction auxiliary agent, organic molecule is in the environment of repulsion, and in time being undertaken extracting by extraction agent hereafter, free organic molecule is easier to be entered in extraction agent from these tissue samples, thus reaches and be separated completely.The organic molecule separated from tissue such as more than at least 60% enters in extraction agent, and this ratio can be at least 70%, 80% or 90%, more than 95%.
extraction agent
In the extraction process of reality, first can carry out the process of extraction auxiliary agent to sample, and then add extraction agent; Also extraction auxiliary agent and extraction agent can be joined in sample simultaneously, two kinds of effects are carried out simultaneously.
According to the composition of material, generally extraction agent is divided into, 1. arene, as (poisonous) such as benzene,toluene,xylenes; 2. fat hydrocarbon: pentane, hexane, octane etc. (low toxicity); 3. alicyclic hydrocarbon type, as (low toxicities) such as cyclohexane, cyclohexanone, toluene cyclohexanone; 4. halogenated hydrocarbons, as (poisonous) such as chlorobenzene, dichloro-benzenes, methylene chloride; 5. alcohols, as (low toxicities) such as methyl alcohol, ethanol, isopropyl alcohols; 6. ethers: as ether, epoxypropane etc. (poisonous); 7. ester class: as (low toxicities) such as methyl acetate, ethyl acetate, propyl acetate, butyl acetates; 8. ketone: as acetone, espeleton, methylisobutylketone etc. (easily system is quick-fried, by control); 9. diol, derivatives: as (poisonous) such as glycol monoethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ethers;
other: are as (poisonous) such as acetonitrile, pyridine, phenol.These extraction agent can use as extraction agent above.
Generally, when selecting suitable extraction agent, the extraction agent selected is allowed preferably to possess such characteristic: the similarity degree of the interesting small organic molecules under the polarity of this extraction agent and non-electriferous state is higher than sample and extract auxiliary agent.Certainly, as a preferred mode, this extraction agent or partial extraction reagent and sample and extract auxiliary agent and can not mix, to ensure layering (or phase-splitting), can better be separated like this.As a preferred mode, this extraction agent possesses low toxicity or nontoxic, chemical inertness, and without the quick-fried toxicity matter processed of system, can under any circumstance can operate like this, and safer and easy.In addition, supplement as preferred, at normal temperatures and pressures, extraction agent is liquid, and low-viscosity.Meeting above-mentioned characteristic extraction reagent in the present invention, can be ester class, nitrile, and the potpourri of ester class and alcohols or ethers or nitrile.
In the present invention, extraction agent preferably with sample with extract auxiliary agent and can be separated, such as naturally layering or by conventional mechanical effect, to be such as centrifugally separated.Such as, due to the low pole feature of extraction agent, and the cohesion of biomacromolecule and the multidigit point effect greatly between molecule in sample, in standing long enough time situation, extraction agent and extraction auxiliary agent and sample will natural separation, show as layering in appearance, and layering interfaces are obvious.But in actual applications, extracting to better carry out the efficiency improving extraction, also can adopt centrifugal method.Namely, when certain centrifugal force, immiscible each phase quick separating is made.General separating effect is determined by the product of centrifugal force and centrifugation time.In low centrifugal force situation, out of phase separation can be realized by extending centrifugation time, in high centrifugal force situation, can separation be completed at short notice.For the present invention, centrifugal force can be more than 1000g, and centrifugation time is set in less than 20 minutes.
nonpolar adjustment reagent and immune response compatibilizing agent
In the present invention, when analyte is low concentration, as 10
-7mol/kg sample 10
-10mol/kg sample or following.Under this kind of concentration, the solubleness of analyte is decided by that polarity or the ionization characteristics of analyte itself, the environment of this kind of feature and its existence have relativity to a certain degree.In that case, add in isolated extraction agent can be miscible with extraction agent nonpolar adjustment reagent, or add immune response compatible solutions simultaneously, the solubleness of analyte in extraction agent can be changed like this.In that case, analyte in extraction/non-polar solvent blended liquid phase, will be redistributed with immune response compatible solutions.
A kind of situation is the character not changing analyte itself.Extraction/nonpolar adjustment reagent mixed liquor shows as low pole, strong polarity during immune response compatible solutions shows as, now, the Small molecular of low concentration will be redistributed according to a certain percentage in two, by regulating the extraction of different proportion and the ratio of nonpolar adjustment reagent, and select the buffer solution containing suitable organic solvent can realize this kind of process.By the method for centrifuging, finally get rid of extraction/non-polar solvent.
The character of another kind of situation for a change analyte itself, as carboxylic analyte, immune response compatible solutions is meta-alkali solution, then Small molecular is with anionic state, is re-assigned in immune response compatible solutions; Otherwise for the analyte containing amino, immune response compatible solutions is slant acidity solution, then Small molecular is re-assigned in immune response compatible solutions with cationic state.
In the present invention, nonpolar correctives mainly refers to non-polar solvent.Should at least possess in following characteristics selecting this kind of solvent one or more: the similarity degree of the interesting small organic molecules under the polarity of this non-polar solvent and non-electriferous state is lower than extraction agent.In some preferred modes, this non-polar solvent and extraction agent miscible, and miscible liquids and immune response compatible solvent are not dissolved each other, can natural layering.In other preferred modes, this is nonpolar possesses low toxicity or nontoxic, chemical inertness, and without the quick-fried toxicity matter processed of system.In more preferred mode, at normal temperatures and pressures, this kind of material is in a liquid state nonpolar correctives, and the feature such as low viscosity.Nonpolar correctives can comprise hydro carbons, and corresponding derivant; Or fat hydrocarbon, and corresponding derivant.Nonpolar correctives can include but not limited to pentane, hexane, octane cyclohexane, cyclohexanone, toluene cyclohexanone, tridecane, dodecane, sherwood oil, decahydronaphthalene, terebinthina, gasoline, kerosene etc.
Nonpolar correctives can comprise vegetable oil, the oil squeezed from vegetable seeds or fruit or extract all using as nonpolar correctives, such as soya-bean oil, sesame oil, tung oil, castor oil, coconut oil, safflower oil, sunflower oil, camellia seed oil, maize germ, canola, olive oil and blending stock etc.
immune response intermiscibility reagent
In the present invention, immune response compatibilizing agent is an experimental concept, not obviously immune response is affected as long as meet, in this kind of solution, can specific recognition be there is in antigen and antibody, in conjunction with, and do not have influence on involved biological fixation effect, any one solution weakened of the tracer signal related to through this solution.Such reagent can be include, but are not limited to, phosphate buffered solution, citric acid solution, carbonate buffer solution, MES, TRIS, HEPES, MOPES, physiological saline.
immunology detection
So-called immunology detection is the high sensitivity based on antigen-antibody, the feature of high selectivity association reaction, and the method for detecting antibody or antibody identifiable design antigen or similar structures compound developed.
Antibody (antibody): body antigenic substance stimulate under, immunoglobulin (Ig) that the thick liquid cell be divided into by B cell produces, that can react with corresponding antigens generation specific binding.All antibody is all immunoglobulin (Ig).Utilize antigen to the repetitious stimulation of animal, collect serum, or prepare monoclonal antibody by monoclonal technigue, can obtain the antibody of specific recognition antigen, namely under suitable solution environmental, finite concentration antigen can by antibody recognition, in conjunction with, this kind of concentration is low especially, generally all 10
-12under mol/L level, this identification simultaneously possesses high specificity, such as, identify antibody epitope facing the benzene ring structure of a nitro, can not identify the benzene ring structure of interband position nitro under comparable sodium or the condition exceeding thousands of times of concentration.Utilize this kind of feature, the immunology detection technology based on antigen-antibody reaction grows up from eighties of last century fifties, and in medical science, microbiological Test aspect progressively starts commercial applications.
The essence of antibody is protein, applies the character of its amino acid residue and protein itself, antibody can be combined or be fixed on certain polymer surface with various probe material, for detectable antigens material.These probe materials comprise collaurum, enzyme, radiomaterial, fluorescent dye, and fixing macromolecular material comprises nitrocellulose filter, polystyrene, Polyvinylchloride, tygon, nylon etc.
In concrete application, for various probe material, develop the detection method with different performance respectively, as colloidal gold technique.Gold immunological technique is a kind of immunolabelling technique using collaurum as label, collaurum refers to that golden fine particle (1-100nm) is dispersed in the system formed in another kind of material, be often referred to the aurosol that the dispersion of golden fine particle is formed in the solution, with this aurosol labelled protein (antigen, antibody or SPA, SPG), colloid gold particle has the characteristic of high electron density, therefore in the antigen-antibody junction of gold mark albumen, visible pitchy particle under microscope; When these labels are assembled in a large number in corresponding mark, naked eyes red color visible or pink spot can be presented on carrier macromolecular material, thus for the location or qualitative of antigen or antibody materials.The device containing immunoreagent bar is utilized to detect in sample whether in a lot of prior art, have announcement or description containing analyte.These reagent strips generally comprise reagent areas and surveyed area, and wherein reagent areas can comprise sample application zone and marked region.Surveyed area comprises the region of display testing result and is positioned at the testing result control zone in surveyed area downstream.Usually, the sample application zone of these reagent strips comprises sample blank film, marked region comprises marker slip, these two regions form reagent areas jointly, reagent areas has comprised the necessary reagent of reaction, surveyed area comprises detection lug, detection lug comprises the region shown test results and the testing result being positioned at this region downstream control control zone, general testing result region is linear, and on testing result region, Show Color change represents in sample, whether analyte exists; Suction zone comprises water absorbent sheet, and these parts join end to end and allow liquid can flow to the other end from one end of reagent strip along the direction of arrow indication to complete test.General conventional reagent strip is nitrocellulose assay strip, and namely surveyed area comprises nitrocellulose filter, nitrocellulose filter is fixed specific binding molecule to show the result of detection; Can also be cellulose acetate membrane or nylon membrane etc.The reagent strip that such as the following patent describes or the device containing reagent strip: US4857453; US5073484; US5119831; US5185127; US5275785; US5416000; US5504013; US5602040; US5622871; US5654162; US5656503; US5686315; US5766961; US5770460; US5916815; US5976895; US6248598; US6140136; US6187269; US6187598; US6228660; US6235241; US6306642; US6352862; US6372515; US6379620; And US6403383.
For Enzyme-multiplied immune technique, by antigen or antibody labeling enzyme, antibody corresponding with it and antigen or antigen and antibody are fixed on macromolecular material, the antigen in liquid or antibody free diffusing is made under liquid environment, when containing tested substance in liquid, this material will have influence on the antigen of mark or the reaction of antibody and fixing antigen or antibody, by washing, by separating in conjunction with the antigen being labeled enzyme of state or antibody and free state, reaction is touched by enzyme, amplifying signal, the power of comparison signal, whether reflect in detected liquid containing target antigen.This kind of method relative to the highly sensitive several times of colloidal gold method, and passes through the typical curve of Offered target antigen or antibody, can accomplish quantitative measurement target substance.As above two kinds of methods all do not need expensive experimental facilities to complete, and relative to mass spectrum, the instrumental methods such as chromatogram have incomparable cost and can operational advantage.
Organic molecule medicine generally can not independently become the antigen causing organism immune response, and specialty claims this kind of material to be haptens.When with carrier covalent bond after, can as holoantigen, in order to Dispersal risk.Due to the high sensitivity of immunological response, with high specificity, and in the widespread use of field of medical examination, correlation technique is comparatively ripe.And based on the various technology of same principle, progressively permeated to the mensuration of small-molecule drug, especially, recently carry out gradually in field of detection of food safety application.
Immunology detection can be comprise enzyme linked immunosorbent assay, collaurum emulsion process, immune agglutination method, radioimmunoassays, immune affine series of strata technology, immunofluorescence technique, and the method such as immunochemiluminescence.
Embodiment
In order to the present invention will be described in detail further, existing citing is described, and these explanations just carry out limited enumerating under marrow of the present invention, can not make any restriction to the scope of claim of the present invention.
Embodiment 1: use principle involved in the present invention to utilize enzyme linked immunological kit to measure pork, pork liver, beef, in the analog sample of beef liver tissue, Clenbuterol molecule is residual.
The preparation of sample: at beef, pork, beef liver, adding forbidden drugs Clenbuterol molecule (being commonly called as " clenbuterol hydrochloride ") in pork liver sample, is 0.5ug/Kg to its content, in order to Reality simulation sample, the sample adding clenbuterol hydrochloride is left standstill 48 hours at 4-8 DEG C.
The solvent that pre-treatment uses comprises:
Extraction auxiliary agent: concentration is the sodium hydrate aqueous solution of 0.1-10M;
Extraction agent: ethyl acetate;
Nonpolar correctives: heptane;
Immune response compatible solutions: pH is the phosphate buffer solution of 6.0-7.0.
The concrete implementation and operation of pre-treatment:
Accurately take 1g tissue samples, add NaOH (extraction auxiliary agent) 1mL, add ethyl acetate (extraction agent) 1mL, quick oscillation is after 10 minutes, is under the condition of 4000g centrifugal 5 minutes at centrifugal force;
The ratio of to be the phosphate buffer solution (immune response compatible solutions) of 6.0-7.0 and heptane (nonpolar correctives) by supernatant liquid and pH with volume ratio be 2:1:4 mixes, and leaves standstill or centrifugal after layering, takes off layer liquid and carries out immunoassays.The immune detection carried out is as follows:
(1), with commercially available Clenbuterol enzyme linked immunological kit (r-biopharm,
clenbuterolFast, Lot:12280), the sensitivity of its kit is 0.1ppb.
Result:
Embodiment 2: according to commercially available Clenbuterol enzyme linked immunological kit (r-biopharm,
clenbuterolFast, Lot:
12280) instructions carried on carries out method of operating and carries out Sample pretreatment and carry out Clenbuterol molecule, and concrete method is specific as follows:
Take sample 5g sample in embodiment 1 in centrifuge tube, add the Tris damping fluid of 25ml50mM, homogenate 30 minutes; Add 15mL normal hexane, and vibrate 5 minutes;
At 10-15 DEG C, take centrifugal force as centrifugal 5 minutes of 4000g
Re-use normal hexane grease removal once
Add 0.5mL5-6M hydrochloric acid to vibrate 1 hour, or spend the night;
Take 6g(and be equivalent to 1g sample) with centrifuge tube;
At 10-15 DEG C, take centrifugal force as centrifugal 5 minutes of 4000g;
Supernatant is moved in another centrifuge tube, add 300 μ L NaOH, and mix 15 minutes.
Add 4mL500mM potassium phosphate buffer PH3.0,4 DEG C of mixing 1.5 hours
At 10-15 DEG C, take centrifugal force as centrifugal 5 minutes of 4000g
Supernatant is used
c18 crosses post.
First use the drip washing of 3mL methyl alcohol
Use 50mM potassium phosphate buffer PH3.0 drip washing again
Add sample
Add 50mM potassium phosphate buffer PH3.0 drip washing again
Vacuum filtration method is used to drain
Use methanol-eluted fractions again
Collect eluent, blow at 50-60 DEG C of nitrogen
Dissolve with 0.4mL pure water, to be measured.
In order to do horizontal contrast with the present invention, add 1.6mL pure water again by sample, to reach and identical extension rate.Result is as follows:
By as above implementing the contrast of 1.2, the method simple and fast that the present invention uses, under the same conditions, with traditional to need the kit of complex steps pre-treatment to carry methods and results suitable.Complete whole mensuration, the present invention only can need complete at 1 hours, does not relate to Nitrogen evaporator, vacuum pump, the experiment equipments such as decontaminating column; And at present commercial reagent cassette method not only needs experiments supporting equipment, and step is complicated, consuming timely at least wants more than 6 hours.
Embodiment 3: commercially available Clenbuterol horizontal immunoreagent bar detection kit (nine ancient cooking vessels are biological, SJJ1) is to carrying out processing to sample by mode of the present invention and carrying comparing of the testing result to sample processing method recorded in instructions.
reagent of the present invention processes
The preparation of sample: adding forbidden drugs Clenbuterol molecule (being commonly called as " clenbuterol hydrochloride ") in pork sample, is 3ug/Kg to its content, in order to Reality simulation sample, leaves standstill 48 hours by the sample adding clenbuterol hydrochloride at 4-8 DEG C.
Disposal route is shown in the method in embodiment 1, is specially:
Accurately take 1g tissue samples, add NaOH (extraction auxiliary agent) 1mL, add ethyl acetate (extraction agent) 1mL, quick oscillation is after 10 minutes, is under the condition of 4000g centrifugal 5 minutes at centrifugal force;
The ratio of to be the phosphate buffer solution (immune response compatible solutions) of 6.0-7.0 and heptane (nonpolar correctives) by upper strata and pH with volume ratio be 1:1:4 mixes, and leaves standstill or centrifugal after layering, takes off layer liquid and carries out immunoassays.
Concrete outcome is as follows:
| Sample | Negative number | Positive number | Invalid |
| Blank 20 parts, pork sample | 20 | 0 | 0 |
| 20 parts, 3ug/Kg pork sample | 0 | 20 | 0 |
Inefficiency: 0/40 × 100%=0%
False negative rate: 0/20 × 100%=0%
False positive rate: 0/20 × 100%=0%.
the instructions carried according to (nine ancient cooking vessels are biological, SJJ1) in commercially available Clenbuterol horizontal immunoreagent bar detection kit enters sample row relax, specific as follows:
By 20 grams same, pork sample, load in the centrifuge tube of 50ml, tighten pipe lid (not allowing water enter in pipe).Centrifuge tube heating water bath 10-15 minute more than 90 DEG C of sample will be housed, and take out and be cooled to room temperature, and get transudate and measure.
The concrete outcome measured after application specifications operation is as follows:
| Sample | Negative number | Positive number | Invalid |
| Blank 20 parts, pork sample | 17 | 2 | 1 |
| 20 parts, 3ug/Kg pork sample | 4 | 14 | 2 |
Inefficiency: 3/40 × 100%=7.5%
False negative rate: 4/18 × 100%=22%
False positive rate: 2/19 × 100%=10.5%
By as above contrasting, method provided by the invention can effectively extract material to be checked, eliminates matrix effect simultaneously.Although the method that test strips instructions carries looks simple, gentleness be added and rise again consuming time also quite long, effectively can not remove matrix effect simultaneously, make generation a certain proportion of invalid, false negative and false positive results.
Embodiment 4: use principle involved in the present invention to utilize enzyme linked immunological kit to measure pork, pork liver, beef, in the analog sample of beef liver tissue, Ractopamine molecule is residual.
The preparation of sample: at beef, pork, beef liver, adds forbidden drugs Ractopamine molecule in pork liver sample, be 0.5ug/Kg to its content, in order to Reality simulation sample, the sample adding clenbuterol hydrochloride is left standstill 48 hours at 4-8 DEG C.
The solvent used comprises:
Extraction auxiliary agent: concentration is the NaOH of 0.1-10M and the aqueous solution of 1-20% sodium sulphate;
Extraction agent: ethylacetate/acetonitrile mixed solution (ethyl acetate: the ratio of acetonitrile is from 4:1 to 9:1);
Nonpolar correctives: sherwood oil;
Immune response compatible solutions: pH is the citric acid solution of 5.5-6.5.
Concrete disposal route is as follows:
Accurately take 1g tissue samples, add the NaOH of 0.1-10M and (extraction auxiliary agent) 1mL of 1-20% sodium sulphate, add ethylacetate/acetonitrile mixed solution (extraction agent) 1mL, quick oscillation is after 5 minutes, is under the condition of 4000g centrifugal 5 minutes at centrifugal force;
The ratio of to be the citric acid solution (immune response compatible solutions) of 5.5-6.5 and sherwood oil (nonpolar correctives) by upper solution and pH with volume ratio be 1:1:3 mixes, and leaves standstill or centrifugal after layering, takes off a layer liquid assay.
The commercially available Ractopamine enzyme linked immunological kit that uses (Beijing Qin Bang Bioisystech Co., Ltd, RACT-CA055, lot:B-110413-B10), the sensitivity of its kit is 0.1ppb.
Result:
(2), same sample, the instructions method carried by commercially available Ractopamine enzyme linked immunological kit (Beijing Qin Bang Bioisystech Co., Ltd, RACT-CA055, lot:B-110413-B10) processes sample, specific as follows:
Get 2g sample to join in 50mL centrifuge tube, add 8mL and extract working fluid, vibrate 10 minutes, centrifugal 10 minutes of 3000g, get supernatant and measure.
The result measured is:
As above data show, the difference that the pre-treating method that by specification operates can not be distinguished dummy significantly and contain between 0.5 μ g/Kg Rct opamine residue sample.Method of the present invention is adopted then obviously to increase difference, in fact need not by instrument, naked eyes can be distinguished.In order to reduce operator in practical operation, instrument can be coordinated to read data, make result have more objectivity.
Claims (15)
1. extract a reagent for analyte in sample, this reagent comprises:
Extraction auxiliary agent, is allowed by this extraction auxiliary agent and is in analyte in sample and this sample separation, or/and allow analyte be non-band electrical property, or/and in the environment allowing analyte be in be ostracised;
Extraction agent, allows analyte be extracted in extraction agent from the environment after being extracted auxiliary agent process by this extraction agent;
Wherein, described reagent also comprises nonpolar adjustment reagent, and this nonpolar adjustment reagent and extraction agent can miscible formation miscible liquids; Wherein, described reagent also comprises immune response intermiscibility reagent, and the miscible liquids of this immune response compatible solutions and nonpolar adjustment reagent and the miscible formation of extraction agent is immiscible.
2. reagent according to claim 1, wherein, extraction agent is organic extraction reagent, and the similarity degree of the analyte under the polarity of extraction agent and non-electriferous state is higher than sample and extract auxiliary agent.
3. reagent according to claim 1, wherein, this extraction agent or partial extraction reagent and sample and to extract auxiliary agent not miscible.
4. reagent according to claim 1, wherein, the similarity degree of the analyte under the polarity of this nonpolar adjustment reagent and non-electriferous state is lower than extraction agent.
5. reagent according to claim 1, the volume ratio of extraction agent and nonpolar correctives is 0.2-10.
6. reagent according to claim 1, the volume ratio of extraction agent and immune response intermiscibility reagent is 0.2-2.
7. according to the reagent one of claim 1-6 Suo Shu, wherein, extract auxiliary agent and comprise the inorganic salts being greater than 20% the solubleness of 10-50 DEG C with water.
8. according to the reagent one of claim 1-6 Suo Shu, wherein, extraction agent is-, ester class, nitrile, alcohols, ethers, and the potpourri of ester class and nitrile, alcohols or ethers.
9. according to the reagent one of claim 1-6 Suo Shu, wherein, nonpolar correctives is varsol or vegetable oil.
10. reagent according to claim 1, wherein, immune response intermiscibility reagent comprises phosphate buffered solution, citric acid solution, carbonate buffer solution, one or more in MES, TRIS, HEPES, MOPES or physiological saline.
11. reagent according to claim 1, wherein, analyte comprises beta-2-agonists class medicine, chloromycetin, sulfa drugs, tetracycline, nitrofurans metabolin, fluoroquinolones, beta-lactam antibiotic, malachite green, concealed malachite green, crystal violet, melamine, olaquindox, 19-nortestosterone, Trenbolone, norethindrone, stilbestrol, Florfenicol, medroxyprogesterone acetate, estradiol or stable in one or more.
12. 1 kinds of methods extracting analyte from sample, the method comprises the following steps:
(1), allow extraction auxiliary agent and sample contact, this extraction auxiliary agent is by the analyte in sample and sample separation; And/or allow analyte be non-band electrical property, or/and in the environment allowing analyte be in be ostracised;
(2), allow sample contact with extraction agent, allow analyte be extracted in extraction agent;
Wherein, the method is also included in extraction agent and adds nonpolar adjustment reagent, allows nonpolar adjustment reagent and extraction agent can miscible formation miscible liquids; Wherein, the method also comprises the miscible liquids allowing described nonpolar adjustment reagent and extraction agent be formed and mixes with immune response intermiscibility Reagent evaluation.
13. methods according to claim 12, allow sample first contact with extraction auxiliary agent, and then allow sample contact with extraction agent; Or, allow the while of extracting auxiliary agent and extraction agent and sample contact; Or, allow sample first contact with extraction agent, and then allow sample contact with extraction auxiliary agent with the potpourri of extraction agent.
14. methods according to claim 12, allow the similarity degree of the analyte under the polarity of extraction agent and non-electriferous state higher than sample and extract auxiliary agent.
15. 1 kinds of methods extracting analyte from sample, the method comprises the following steps:
(1), allow extraction compounding agent solution and sample contact, this extraction auxiliary agent is by the analyte in sample and sample separation; And/or allow analyte be non-band electrical property, or/and in the environment allowing analyte be in be ostracised;
(2), allow sample contact with extraction agent solution, allow analyte be extracted in extraction agent;
Wherein, the mix reagent containing extraction auxiliary agent, extraction agent and analyte in step (2) relief carries out layering, and then allows the upper solution of layering mix with nonpolar adjustment reagent and immunocompatible solution.
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| CN101852720A (en) * | 2010-05-12 | 2010-10-06 | 山东省农业科学院中心实验室 | Method for biosensor to detect ractopamine in pig flesh |
| CN101936984A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method and special chemical luminous immunoassay kit for detecting clenbuterol |
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| CN101852720A (en) * | 2010-05-12 | 2010-10-06 | 山东省农业科学院中心实验室 | Method for biosensor to detect ractopamine in pig flesh |
| CN101936984A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method and special chemical luminous immunoassay kit for detecting clenbuterol |
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Effective date of registration: 20161221 Address after: Hangzhou City, Zhejiang province Yuhang District 311121 Futailu Thailand Street No. 17 Patentee after: HANGZHOU BIOTEST BIOTECH CO., LTD. Address before: 201714 Shanghai city Qingpu District Zhujiajue Road No. 2588 building 862 Sun Island Shen Patentee before: Li Hanqing |