WO2022193861A1 - Solvent substitution-based magnetic solid-phase extraction method - Google Patents
Solvent substitution-based magnetic solid-phase extraction method Download PDFInfo
- Publication number
- WO2022193861A1 WO2022193861A1 PCT/CN2022/074959 CN2022074959W WO2022193861A1 WO 2022193861 A1 WO2022193861 A1 WO 2022193861A1 CN 2022074959 W CN2022074959 W CN 2022074959W WO 2022193861 A1 WO2022193861 A1 WO 2022193861A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- solvent
- target
- phase extraction
- magnetic
- solid phase
- Prior art date
Links
- 239000002904 solvent Substances 0.000 title claims abstract description 142
- 238000002414 normal-phase solid-phase extraction Methods 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 62
- 238000006467 substitution reaction Methods 0.000 title abstract 2
- 239000000463 material Substances 0.000 claims abstract description 49
- 238000001179 sorption measurement Methods 0.000 claims abstract description 34
- 238000000605 extraction Methods 0.000 claims abstract description 22
- 239000013076 target substance Substances 0.000 claims description 32
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000377 silicon dioxide Substances 0.000 claims description 12
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical group CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000000696 magnetic material Substances 0.000 claims description 8
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- 238000004587 chromatography analysis Methods 0.000 claims description 7
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
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- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 3
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- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims description 2
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- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 2
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims 2
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/12—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the preparation of the feed
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3861—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36 using an external stimulus
- B01D15/3885—Using electrical or magnetic means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
Definitions
- the invention relates to a magnetic solid-phase extraction method, in particular to a magnetic solid-phase extraction method for improving the adsorption rate of a magnetic solid-phase extraction material by solvent replacement.
- Magnetic solid-phase extraction technology is a new and environmentally friendly sample pretreatment method developed in recent years. It does not require centrifugation or filtration. It can be easily The target-adsorbed magnetic material can be separated from the sample solution. Compared with traditional sample pretreatment techniques such as solid-phase extraction, solvent extraction, ultrasonic extraction, etc., the application of magnetic solid-phase extraction greatly simplifies the sample pretreatment process, and it is very easy to achieve phase extraction. separation. Therefore, magnetic solid-phase extraction technology plays an increasingly important role in chemical and biological detection.
- the adsorption is generally carried out directly in the extraction solution of the target substance.
- the extraction solution is generally a good solution for the target
- the magnetic material needs to have a strong adsorption force for the target to realize the adsorption process of the target analyte, and the target is in the magnetic material.
- the distribution balance on the surface and in the solvent is not conducive to the adsorption of magnetic materials.
- the target to be extracted is a trace target
- the magnetic solid phase extraction in the prior art will be more difficult and less efficient to adsorb the trace target in a good solvent.
- the purpose of the present invention is to provide a novel magnetic solid phase extraction method. More specifically, the present invention provides a magnetic solid-phase extraction method for effectively improving the adsorption capacity of a magnetic material for a target analyte in a solid-phase extraction process by solvent replacement.
- the inventors found that by replacing the good solvent in the extraction solution containing the target with a poor solvent for the target, the partition coefficient of the target in the extraction solution and the solid phase (ie, the magnetic solid-phase extraction material) was changed, thereby The partition coefficient of the target in the extraction solution is reduced, the extraction equilibrium is shifted to the solid phase, and the adsorption rate of the solid to the target is improved.
- the present invention has been obtained.
- the present invention provides a solid phase extraction method, comprising:
- step c) adding a magnetic solid phase extraction material to the second solution obtained in step b) to adsorb the target substance.
- the present invention provides a method for detecting whether a target exists in a sample to be tested, the method comprising implementing the magnetic solid phase extraction method described in the first aspect.
- the present invention provides an improved magnetic solid phase extraction method, which can effectively improve the magnetic solid phase extraction by replacing the good solvent in the extraction solution containing the target substance with the poor solvent of the target substance, and then performing magnetic solid phase extraction.
- the adsorption capacity of the extraction material to the target substance can realize the effective separation of the target substance from the extraction solution, so that the magnetic solid-phase extraction method of the present invention is particularly suitable for the solid-phase extraction of trace target substances, and further enables the method of the present invention to be used in chemical biology.
- Chemical detection such as immunodetection, chromatographic analysis, etc. have been more widely and effectively used.
- the inventors improved the magnetic solid-phase extraction method by replacing the good solvent in the extraction solution containing the target with the poor solvent of the target, and improved the adsorption rate of the magnetic solid-phase extraction material to the target.
- the target substance can be more effectively separated from the extraction solution, so that the magnetic solid-phase extraction method of the present invention is not only particularly suitable for solid-phase extraction of trace target substances, but also can be more widely and effectively applied to chemical biological detection such as immunoassay. detection, chromatographic analysis, etc.
- the present invention provides a magnetic solid phase extraction method, comprising:
- step c) adding a magnetic solid phase extraction material to the second solution obtained in step b) to adsorb the target substance.
- the first solvent is a good solvent for the target, and can be used to extract the target from the sample to be tested in the present invention, that is, the extraction solvent for the target.
- the second solvent is a poor solvent for the target, and is used in the present invention as an adsorption solvent for the target, that is, a solvent that facilitates the adsorption of the target to the magnetic solid phase extraction material.
- the so-called “good solvent” and “poor solvent” refer to the solubility of the target substance therein, and are a relative concept.
- the “poor solvent” of the target refers to a solvent having a weaker dissolving ability for the target than the "good solvent” of the target.
- the “good solvent” of the target refers to a solvent that has a stronger ability to dissolve the target than the “poor solvent” of the target.
- the equilibrium is meant the equilibrium between the distribution of the target substance between the solvent and the solid surface.
- the solvent is a good solvent, the equilibrium will tend to the solvent; on the contrary, if the solvent is a poor solvent, the equilibrium will tend to the surface of the magnetic solid phase material.
- the "equilibrium” here is a dynamic equilibrium, as the dissolved part of the target in the poor solvent is adsorbed by the magnetic solid-phase extraction material, the insoluble part of the target is further dissolved in the poor solvent, and passed through The equilibrium is bound to the magnetic solid-phase extraction material, thereby effectively enhancing the adsorption of the target species by the magnetic solid-phase extraction material.
- the method further comprises: prior to performing step b), the first solvent is evaporated to dryness.
- Vaporizing the first solvent to dryness provides many benefits.
- impurities will slowly precipitate, and some impurities will be deposited on the surface of the magnetic solid phase extraction material, thereby greatly reducing the efficiency of subsequent elution.
- the precipitated impurities may also cause the aggregation of magnetic solid-phase extraction materials, further reducing the subsequent elution efficiency.
- the selection of the first solvent as the extraction solvent for the target can be more diverse, without considering the interference of the first solvent on the magnetic solid-phase extraction adsorption process, Thereby, the extraction efficiency can be maximized.
- the first solvent is evaporated by heat and/or gas flow, and/or vacuum.
- the specific conditions for drying out can be selected empirically by those skilled in the art, which are within their capabilities.
- the volume of the second solvent is the same as or different from the volume of the first solvent.
- the volume of the second solvent is less than the volume of the first solvent. Evaporating the first solvent to dryness and then adding the second solvent less than the volume of the first solvent can improve the interaction probability between the surface of the magnetic solid phase extraction material and the target, and further improve the adsorption of the magnetic solid phase extraction material to the target.
- the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 50%.
- the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 70%.
- the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 90%.
- the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 99%.
- the magnetic solid phase extraction method further comprises: after step c), first separating the second solution and the magnetic solid phase extraction material, and then using a third solvent to separate the magnetic solid phase extraction material from the magnetic solid phase extraction material.
- the target substance is eluted from the solid phase extraction material to obtain a third solution containing the target substance, wherein the solubility of the target substance in the third solvent is higher than that in the second solvent .
- the third solvent used for eluting the target substance adsorbed on the magnetic solid phase extraction material is the same as the first solvent. It is also a good solvent for the target.
- the adsorption rate of the magnetic solid phase extraction material to the target in the second solvent is higher than that in the first solvent and/or the third solvent.
- the adsorption rate of the material to the target can be better adsorbed by the magnetic solid phase extraction material.
- the magnetic solid phase extraction method further comprises: washing the magnetic solid phase extraction material with a fourth solvent before eluting the target from the magnetic solid phase extraction material with a third solvent Phase extraction material.
- the purpose of the washing step is to remove impurities that may be non-specifically adsorbed to the magnetic SPE material before the target is eluted.
- the fourth solvent may be a poor solvent for the target.
- the solubility of the target substance in the fourth solvent used in the washing step should be much lower than that in the third solvent used in the elution step, for example, a maximum of only It is 50% of its solubility in the third solvent used for the elution step, preferably up to only 30%, more preferably up to only 10%. Most preferably, the target is completely insoluble in the fourth solvent used in the washing step.
- both the first solvent and the third solvent are good solvents for the target. Therefore, in yet another specific embodiment, the first solvent and the third solvent are the same or different, and can be independently selected from C 1 -C 6 monohydric alcohols, C 1 -C 6 polyhydric alcohols, Acetonitrile, ethyl acetate, substituted methane, acetone, benzene or substituted benzene, or any combination thereof, but not limited thereto.
- the C 1 -C 6 monohydric alcohol may be methanol, ethanol or propanol, etc., but is not limited thereto.
- the C 1 -C 6 polyol may be ethylene glycol or propylene glycol, etc., but is not limited thereto.
- the substituted methane may be dichloromethane, trichloromethane, tetrachloromethane, etc., but is not limited thereto.
- the substituted benzene may be toluene, xylene, etc., but is not limited thereto.
- the first solvent is doped with an acid, such as an organic or inorganic acid; or a base, such as an organic or inorganic base.
- the organic acid may be trifluoroacetic acid, trichloroacetic acid, formic acid, acetic acid, propionic acid, or p-toluenesulfonic acid, but is not limited thereto.
- the inorganic acid may be hydrochloric acid, nitric acid, or sulfuric acid, but is not limited thereto.
- the organic base may be triethylamine, tripropylamine, or methylamine, but is not limited thereto.
- the inorganic base may be sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, or disodium hydrogen phosphate, but is not limited thereto.
- both the second solvent and the fourth solvent are poor solvents for the target.
- the second solvent and the fourth solvent may be the same or different, and may each be independently selected from liquid alkanes, ethers, or any combination thereof.
- the liquid alkane may be n-hexane, cyclohexane, etc., but is not limited thereto.
- the ethers can be petroleum ether, diethyl ether, etc., but are not limited thereto.
- the magnetic solid phase extraction material is a magnetic nanomaterial, such as but not limited to magnetic silica nanoparticles, magnetic polymer nanoparticles, magnetic fullerene nanoparticles, and the like.
- the target is a trace target.
- the term "trace amount”, as understood by a person skilled in the art, refers to a very small amount, eg an amount below one part per million. Due to its extremely low content, the adsorption of trace targets in good solvents by solid-phase extraction materials will also be more difficult and less efficient. The trace target can be well dispersed in the poor solvent, thereby promoting the adsorption of the trace target by the solid phase extraction material. Therefore, the method of the present invention is particularly suitable for solid phase extraction of trace targets.
- the target can be a small molecule compound, nucleic acid, phospholipid, and protein or polypeptide.
- small molecule compound refers to a compound with a very small molecular weight, usually a compound with a molecular weight of less than 1000 Daltons, especially a molecular weight of less than 500 Daltons.
- the small molecule compound can be pesticide residues, such as but not limited to pesticides, fungicides, algaecides, insecticides, defoliants; veterinary drug residues, such as but not limited to antibiotics, Sulfonamides, furans, antiparasitic drugs, hormone drugs, etc.; mycotoxins, such as but not limited to aflatoxins; marine toxins, such as but not limited to tetrodotoxin, cigatoxin, etc.; illegal food additives, such as but not limited to Bleach, Sudan red, melamine, etc.; heavy metal residues, such as but not limited to mercury, lead, cadmium, etc.
- pesticide residues such as but not limited to pesticides, fungicides, algaecides, insecticides, defoliants
- veterinary drug residues such as but not limited to antibiotics, Sulfonamides, furans, antiparasitic drugs, hormone drugs, etc.
- mycotoxins such as but not limited to aflatoxins
- marine toxins such
- the nucleic acid may be DNA or RNA.
- the protein or polypeptide may be casein, whey protein, glucan phosphorylase, cytochrome C oxidase, N-glycoprotein fumarate, etc., but not limited thereto.
- the sample to be tested is food or feed.
- the food can be dairy products, vegetables such as potatoes, tomatoes, etc., fruits such as sugar cane, apples, pears, etc., aquatic products such as fish, shrimp, shellfish, etc., meats such as pork, beef, Chicken, etc., grain and oil products such as wheat, flour, black rice, rapeseed oil, soybean oil, etc., health food such as capsules, powder, etc., but not limited to this.
- the feed may be animal feed such as poultry, aquatic products, etc., but is not limited thereto.
- the present invention provides a method for detecting whether a target exists in a sample to be tested, the method comprising implementing the magnetic solid phase extraction method described in the first aspect.
- the second aspect of the present invention includes the various features and advantages described above with respect to the method of the first aspect of the present invention, which will not be repeated here.
- the method further comprises performing an immunodetection step or a chromatographic analysis step.
- the immunodetection can be lateral flow chromatography, enzyme-linked immunosorbent assay, or chemiluminescence, but is not limited thereto.
- the chromatographic analysis can be high performance liquid chromatography, liquid chromatography-mass spectrometry, or gas-mass spectrometry detection, but is not limited thereto.
- the magnetic silica nanospheres were adsorbed to the tube wall with a magnet, and then the solution was removed. The magnet was removed again, 500 ⁇ L of 20% methanol in Tris-HCl buffer (pH 7.5, 100 mM) was added as eluent, and vortexed for 1 minute. The magnetic silica nanospheres were again adsorbed on the tube wall with a magnet, and 200 ⁇ L of the eluate was drawn, added to the colloidal gold container containing the conjugated chloramphenicol antibody, and reacted at 35°C for 3 minutes. Chloramphenicol test strips were added, and the reaction was continued for 3 minutes, and then the results were judged by colorimetry (ie, T/C line chromaticity ratio on immunochromatography). The results are listed in Table 1 below.
- the magnetic silica nanospheres were adsorbed on the tube wall with a magnet, and then the solution was pipetted. The solution was filtered with a 0.22 ⁇ m filter, then measured by LCMS/MS, and the concentration of aflatoxin B1 in the solution was calculated, thereby calculating the effect of magnetic silica nanospheres on aflatoxin B1 in samples 1-4.
- the adsorption rate of the results are listed in Table 2 below. Table 2: The concentration of aflatoxin B1 in the adsorbed solution and the adsorption rate of magnetic silica nanospheres
- the magnetic solid phase extraction method by solvent replacement according to the present invention can adsorb the target substance more efficiently, and the adsorption rate is 19.0% higher than that of sample 1-2 (direct method). %-22.3% increased to 89.9%-91.5% of samples 1-4, and the adsorption rate was about 4-6 times that of the direct method.
Abstract
A magnetic solid-phase extraction method for improving the adsorption rate of a magnetic solid-phase extraction material by means of solvent substitution. A solvent in an extraction solution containing a target is substituted with a poor solvent of the target to change a partition coefficient of the target in the extraction solution and a solid phase, so that the partition coefficient of the target in the extraction solution is reduced, an extraction equilibrium tips to the solid phase, and the adsorption rate of the solid phase for the target is improved. The method is applicable to extraction of trace targets.
Description
本发明涉及一种磁性固相萃取方法，具体地涉及一种通过溶剂置换以提高磁性固相萃取材料的吸附率的磁性固相萃取方法。The invention relates to a magnetic solid-phase extraction method, in particular to a magnetic solid-phase extraction method for improving the adsorption rate of a magnetic solid-phase extraction material by solvent replacement.
磁性固相萃取技术是近些年发展起来的一种新型、环境友好的样品前处理方式，不需要离心或过滤，仅通过施加外部磁场，利用磁性材料对于分析物的吸附作用，就可以很容易地从样品溶液中分离吸附了目标物的磁性材料。与传统的样品预处理技术如固相萃取法、溶剂萃取法、超声提取法等相比，磁性固相萃取法的运用在极大程度上简化了样品前处理的过程，并极易实现相的分离。因此，磁性固相萃取技术在化学生物学检测中起到了越来越重要的作用。Magnetic solid-phase extraction technology is a new and environmentally friendly sample pretreatment method developed in recent years. It does not require centrifugation or filtration. It can be easily The target-adsorbed magnetic material can be separated from the sample solution. Compared with traditional sample pretreatment techniques such as solid-phase extraction, solvent extraction, ultrasonic extraction, etc., the application of magnetic solid-phase extraction greatly simplifies the sample pretreatment process, and it is very easy to achieve phase extraction. separation. Therefore, magnetic solid-phase extraction technology plays an increasingly important role in chemical and biological detection.
目前，利用磁性材料进行固相萃取时，一般是在目标物的提取液中直接进行吸附。但是，由于提取液一般是目标物的良溶液，这样在磁性固相萃取过程中，磁性材料需要对目标物具有很强的吸附力才能实现对目标分析物的吸附过程，并且目标物在磁性材料表面及溶剂中的分布平衡不利于磁性材料对其的吸附作用。尤其是，当待萃取的目标物是痕量目标物的情况下，现有技术的磁性固相萃取对于良溶剂中痕量目标物的吸附会更加困难，效率会更低。At present, when magnetic materials are used for solid-phase extraction, the adsorption is generally carried out directly in the extraction solution of the target substance. However, since the extraction solution is generally a good solution for the target, in the magnetic solid phase extraction process, the magnetic material needs to have a strong adsorption force for the target to realize the adsorption process of the target analyte, and the target is in the magnetic material. The distribution balance on the surface and in the solvent is not conducive to the adsorption of magnetic materials. In particular, when the target to be extracted is a trace target, the magnetic solid phase extraction in the prior art will be more difficult and less efficient to adsorb the trace target in a good solvent.
因此，本领域需要一种能够有效提高固相萃取过程中磁性材料对目标分析物的吸附能力的磁性固相萃取方法。Therefore, there is a need in the art for a magnetic solid-phase extraction method that can effectively improve the adsorption capacity of a magnetic material for a target analyte in a solid-phase extraction process.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种新型的磁性固相萃取方法。更具体地，本发明提供一种通过溶剂置换以有效提高固相萃取过程中磁性材料对目标分析物的吸附能力的磁性固相萃取方法。The purpose of the present invention is to provide a novel magnetic solid phase extraction method. More specifically, the present invention provides a magnetic solid-phase extraction method for effectively improving the adsorption capacity of a magnetic material for a target analyte in a solid-phase extraction process by solvent replacement.
本发明人发现，通过将含有目标物的提取溶液中的良溶剂替换为目标物的不良溶剂，以改变目标物在提取溶液和固相(即，磁性固相萃取材料)中的分配系数，从而使得目标物在提取溶液中的分配系数降低，萃取平衡向固相移动，提高固相对目标物的吸附率。由此，得到了本发明。The inventors found that by replacing the good solvent in the extraction solution containing the target with a poor solvent for the target, the partition coefficient of the target in the extraction solution and the solid phase (ie, the magnetic solid-phase extraction material) was changed, thereby The partition coefficient of the target in the extraction solution is reduced, the extraction equilibrium is shifted to the solid phase, and the adsorption rate of the solid to the target is improved. Thus, the present invention has been obtained.
因此，在第一方面，本发明提供了一种固相萃取方法，其包括：Therefore, in a first aspect, the present invention provides a solid phase extraction method, comprising:
a)使用第一溶剂从待测样本制备含有目标物和所述第一溶剂的第一溶液；a) using a first solvent to prepare a first solution containing the target and the first solvent from the sample to be tested;
b)加入第二溶剂以置换所述第一溶剂，从而获得第二溶液，其中，所述目标物在所述第二溶剂中的溶解度低于在所述第一溶剂中的溶解度；b) adding a second solvent to replace the first solvent, thereby obtaining a second solution, wherein the solubility of the target in the second solvent is lower than the solubility in the first solvent;
c)向步骤b)中得到的第二溶液中加入磁性固相萃取材料以吸附所述目标物。c) adding a magnetic solid phase extraction material to the second solution obtained in step b) to adsorb the target substance.
在第二方面，本发明提供了一种待测样本中是否存在目标物的检测方法，所述方法包括实施第一方面所述的磁性固相萃取方法。In a second aspect, the present invention provides a method for detecting whether a target exists in a sample to be tested, the method comprising implementing the magnetic solid phase extraction method described in the first aspect.
综上，本发明提供了改进的磁性固相萃取方法，其通过将含有目标物的提取溶液中的良溶剂替换为目标物的不良溶剂，再进行磁性固相萃取，能够有效地提升磁性固相萃取材料对目标物的吸附能力，实现目标物从提取溶液的有效分离，使得本发明的磁性固相萃取方法特别适用于痕量目标物的固相萃取，进而使得本发明的方法能够在化学生物学检测如免疫检测、色谱分析等方面得到更广泛且有效的应用。In summary, the present invention provides an improved magnetic solid phase extraction method, which can effectively improve the magnetic solid phase extraction by replacing the good solvent in the extraction solution containing the target substance with the poor solvent of the target substance, and then performing magnetic solid phase extraction. The adsorption capacity of the extraction material to the target substance can realize the effective separation of the target substance from the extraction solution, so that the magnetic solid-phase extraction method of the present invention is particularly suitable for the solid-phase extraction of trace target substances, and further enables the method of the present invention to be used in chemical biology. Chemical detection such as immunodetection, chromatographic analysis, etc. have been more widely and effectively used.
下面对本发明进行详细的描述。需理解，以下描述仅以示例方式来对本发明进行说明，无意于对本发明的范围进行限制，本发明的保护范围以随附权利要求为准。并且，本领域技术人员理解，在不背离本发明的精神和主旨的情况下，可以对本发明技术方案进行修改。The present invention will be described in detail below. It should be understood that the following description is only for illustrating the present invention by way of example, and is not intended to limit the scope of the present invention, and the protection scope of the present invention is subject to the appended claims. Moreover, those skilled in the art understand that the technical solutions of the present invention can be modified without departing from the spirit and spirit of the present invention.
如上所述，本发明人通过将含有目标物的提取溶液中的良溶剂替换 为目标物的不良溶剂，改进了磁性固相萃取方法，提高了磁性固相萃取材料对目标物的吸附率，使目标物能够更有效地从提取溶液中分离，进而使得本发明的磁性固相萃取方法不仅特别适用于痕量目标物的固相萃取，而且能够更广泛且有效地应用于化学生物学检测如免疫检测、色谱分析等方面。As described above, the inventors improved the magnetic solid-phase extraction method by replacing the good solvent in the extraction solution containing the target with the poor solvent of the target, and improved the adsorption rate of the magnetic solid-phase extraction material to the target. The target substance can be more effectively separated from the extraction solution, so that the magnetic solid-phase extraction method of the present invention is not only particularly suitable for solid-phase extraction of trace target substances, but also can be more widely and effectively applied to chemical biological detection such as immunoassay. detection, chromatographic analysis, etc.
因此，在第一方面，本发明提供一种磁性固相萃取方法，其包括：Therefore, in a first aspect, the present invention provides a magnetic solid phase extraction method, comprising:
a)使用第一溶剂从待测样本制备含有目标物和所述第一溶剂的第一溶液；a) using a first solvent to prepare a first solution containing the target and the first solvent from the sample to be tested;
b)加入第二溶剂以置换所述第一溶剂，从而获得第二溶液，其中，所述目标物在所述第二溶剂中的溶解度低于在所述第一溶剂中的溶解度；b) adding a second solvent to replace the first solvent, thereby obtaining a second solution, wherein the solubility of the target in the second solvent is lower than the solubility in the first solvent;
c)向步骤b)中得到的第二溶液中加入磁性固相萃取材料以吸附所述目标物。c) adding a magnetic solid phase extraction material to the second solution obtained in step b) to adsorb the target substance.
所述第一溶剂是目标物的良溶剂，在本发明中可以用于从待测样本提取目标物，即目标物的提取溶剂。所述第二溶剂是目标物的不良溶剂，在本发明中用作目标物的吸附溶剂，即，有助于目标物吸附至磁性固相萃取材料的溶剂。用目标物的不良溶剂对目标物的良溶剂进行溶剂置换后，由于目标物在不良溶剂中的溶解度不佳，使得目标物在不良溶剂与磁性固相萃取材料表面的平衡向磁性固相萃取材料倾斜，进而有效提升磁性固相萃取材料对目标物的吸附。The first solvent is a good solvent for the target, and can be used to extract the target from the sample to be tested in the present invention, that is, the extraction solvent for the target. The second solvent is a poor solvent for the target, and is used in the present invention as an adsorption solvent for the target, that is, a solvent that facilitates the adsorption of the target to the magnetic solid phase extraction material. After replacing the good solvent of the target with the poor solvent of the target, due to the poor solubility of the target in the poor solvent, the equilibrium of the target on the surface of the poor solvent and the magnetic solid phase extraction material is changed to the magnetic solid phase extraction material. Tilt, and then effectively improve the adsorption of the magnetic solid phase extraction material to the target.
应当理解，在本发明的上下文中，所谓“良溶剂”和“不良溶剂”是针对目标物在其中的溶解度而言的，并且是一个相对概念。目标物的“不良溶剂”是指相对于目标物的“良溶剂”对目标物具有较弱溶解能力的溶剂。同理，目标物的“良溶剂”是指相对于目标物的“不良溶剂”对目标物具有较强溶解能力的溶剂。It should be understood that in the context of the present invention, the so-called "good solvent" and "poor solvent" refer to the solubility of the target substance therein, and are a relative concept. The "poor solvent" of the target refers to a solvent having a weaker dissolving ability for the target than the "good solvent" of the target. Similarly, the "good solvent" of the target refers to a solvent that has a stronger ability to dissolve the target than the "poor solvent" of the target.
应当理解，在本发明的上下文中，所谓“平衡”是指目标物在溶剂与固体表面分配之间的平衡。对于目标物而言，如果溶剂是良溶剂，那么平衡就会趋向于溶剂；反之，如果溶剂是不良溶剂，平衡则会趋向于磁性固相材料的表面。此外，应当理解，这里的“平衡”是一种动态平 衡，随着目标物在不良溶剂中的溶解部分被磁性固相萃取材料吸附，目标物的不溶解部分进一步溶解于不良溶剂中，并通过平衡被结合到磁性固相萃取材料上，由此有效提升磁性固相萃取材料对目标物的吸附。It should be understood that, in the context of the present invention, by "equilibrium" is meant the equilibrium between the distribution of the target substance between the solvent and the solid surface. For the target, if the solvent is a good solvent, the equilibrium will tend to the solvent; on the contrary, if the solvent is a poor solvent, the equilibrium will tend to the surface of the magnetic solid phase material. In addition, it should be understood that the "equilibrium" here is a dynamic equilibrium, as the dissolved part of the target in the poor solvent is adsorbed by the magnetic solid-phase extraction material, the insoluble part of the target is further dissolved in the poor solvent, and passed through The equilibrium is bound to the magnetic solid-phase extraction material, thereby effectively enhancing the adsorption of the target species by the magnetic solid-phase extraction material.
在一个具体的实施方案中，所述方法还包括：在进行步骤b)之前，先使所述第一溶剂挥干的步骤。In a specific embodiment, the method further comprises: prior to performing step b), the first solvent is evaporated to dryness.
使所述第一溶剂挥干提供很多益处。Vaporizing the first solvent to dryness provides many benefits.
一方面，在对目标物的提取过程中，与目标物一起溶于所述第一溶剂中的一些杂质由于在所述第二溶剂中溶解度不佳，因此在使所述第一溶剂挥干后，所述第一溶剂中的杂质被析出，并且其后不能很好地溶解于所述第二溶剂中，因此无法进入溶液体系。这种情况下，杂质与磁性固相萃取材料的相互作用几率大大降低，进而降低了杂质在洗脱液中的浓度和在对后续实验的干扰。而如果没有挥干步骤，则第一溶剂中溶解的杂质(至少一部分)则不会经历析出再溶解的过程，因此杂质的去除效率则会较低。同时，在后续加入不良溶剂的过程中，杂质会缓慢析出，并且会有部分杂质沉积于磁性固相萃取材料表面，进而大幅降低后续洗脱的效率。析出的杂质也有可能造成磁性固相萃取材料的聚集，进一步降低后续洗脱效率。On the one hand, in the process of extracting the target substance, some impurities dissolved in the first solvent together with the target substance have poor solubility in the second solvent, so after the first solvent is evaporated to dryness , the impurities in the first solvent are precipitated, and then cannot be dissolved in the second solvent well, so they cannot enter the solution system. In this case, the interaction probability of impurities with the magnetic solid phase extraction material is greatly reduced, thereby reducing the concentration of impurities in the eluent and the interference to subsequent experiments. However, if there is no volatilization step, the impurities (at least a part) dissolved in the first solvent will not undergo the process of precipitation and re-dissolution, so the removal efficiency of impurities will be lower. At the same time, in the subsequent process of adding poor solvent, impurities will slowly precipitate, and some impurities will be deposited on the surface of the magnetic solid phase extraction material, thereby greatly reducing the efficiency of subsequent elution. The precipitated impurities may also cause the aggregation of magnetic solid-phase extraction materials, further reducing the subsequent elution efficiency.
另一方面，由于进行了第一溶剂挥干，对所述第一溶剂作为目标物的提取溶剂的选择可以更多元，而无需考虑所述第一溶剂对磁性固相萃取吸附过程的干扰，由此能够最大化提取效率。On the other hand, due to the evaporation of the first solvent, the selection of the first solvent as the extraction solvent for the target can be more diverse, without considering the interference of the first solvent on the magnetic solid-phase extraction adsorption process, Thereby, the extraction efficiency can be maximized.
在一个进一步具体的实施方案中，通过热和/或气流、和/或真空使所述第一溶剂挥干。本领域技术人员可以根据经验对挥干的具体条件进行选择，这在其能力范围内。In a further specific embodiment, the first solvent is evaporated by heat and/or gas flow, and/or vacuum. The specific conditions for drying out can be selected empirically by those skilled in the art, which are within their capabilities.
在一个具体的实施方案中，所述第二溶剂的体积与所述第一溶剂的体积相同或不同。In a specific embodiment, the volume of the second solvent is the same as or different from the volume of the first solvent.
在一个优选的实施方案中，所述第二溶剂的体积少于所述第一溶剂的体积。对第一溶剂进行挥干，然后添加少于第一溶剂体积的第二溶剂，能够提高磁性固相萃取材料表面与目标物的相互作用概率，进一步提高磁性固相萃取材料对目标物的吸附。In a preferred embodiment, the volume of the second solvent is less than the volume of the first solvent. Evaporating the first solvent to dryness and then adding the second solvent less than the volume of the first solvent can improve the interaction probability between the surface of the magnetic solid phase extraction material and the target, and further improve the adsorption of the magnetic solid phase extraction material to the target.
在又一个具体的实施方案中，在所述第二溶剂中所述磁性固相萃取材料对所述目标物的吸附率为至少50％。In yet another specific embodiment, the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 50%.
在一个优选的实施方案中，在所述第二溶剂中所述磁性固相萃取材料对所述目标物的吸附率为至少70％。In a preferred embodiment, the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 70%.
在一个更优选的实施方案中，在所述第二溶剂中所述磁性固相萃取材料对所述目标物的吸附率为至少90％。In a more preferred embodiment, the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 90%.
在一个进一步更优选的实施方案中，在所述第二溶剂中所述磁性固相萃取材料对所述目标物的吸附率为至少99％。In a further more preferred embodiment, the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 99%.
在一个进一步具体的实施方案中，所述磁性固相萃取方法还包括：在步骤c)之后，先分离所述第二溶液与所述磁性固相萃取材料，然后用第三溶剂从所述磁性固相萃取材料中洗脱所述目标物，以获得含有所述目标物的第三溶液，其中，所述目标物在所述第三溶剂中的溶解度高于在所述第二溶剂中的溶解度。可以理解，相对于作为目标物的不良溶剂的第二溶剂，用于将吸附在所述磁性固相萃取材料上的目标物洗脱下来的所述第三溶剂，同所述第一溶剂一样，也是目标物的良溶剂。In a further specific embodiment, the magnetic solid phase extraction method further comprises: after step c), first separating the second solution and the magnetic solid phase extraction material, and then using a third solvent to separate the magnetic solid phase extraction material from the magnetic solid phase extraction material. The target substance is eluted from the solid phase extraction material to obtain a third solution containing the target substance, wherein the solubility of the target substance in the third solvent is higher than that in the second solvent . It can be understood that, with respect to the second solvent, which is a poor solvent for the target substance, the third solvent used for eluting the target substance adsorbed on the magnetic solid phase extraction material is the same as the first solvent. It is also a good solvent for the target.
在一个具体的实施方案中，在所述第二溶剂中所述磁性固相萃取材料对所述目标物的吸附率高于在所述第一溶剂和/或所述第三溶剂中所述磁性材料对所述目标物的吸附率。由此，在所述第二溶剂中的目标物可以更好地被磁性固相萃取材料所吸附。In a specific embodiment, the adsorption rate of the magnetic solid phase extraction material to the target in the second solvent is higher than that in the first solvent and/or the third solvent. The adsorption rate of the material to the target. Thus, the target in the second solvent can be better adsorbed by the magnetic solid phase extraction material.
在一个更进一步的实施方案中，所述磁性固相萃取方法还包括：在用第三溶剂从所述磁性固相萃取材料中洗脱所述目标物之前，使用第四溶剂洗涤所述磁性固相萃取材料。洗涤步骤的目的是在对目标物进行洗脱之前，将可能非特异性吸附于磁性固相萃取材料的杂质除去。可以理解，与第二溶剂类似，第四溶剂可以是目标物的不良溶剂。同样可以理解，为避免洗涤步骤造成目标物的损失，目标物在用于洗涤步骤的第四溶剂中的溶解度应远低于其在用于洗脱步骤的第三溶剂中的溶解度，例如最高仅为其在用于洗脱步骤的第三溶剂中的溶解度的50％，优选最高仅为30％，更优选最高仅为10％。最优选地，目标物在用于洗涤步骤的第四溶剂中是完全不溶的。In a further embodiment, the magnetic solid phase extraction method further comprises: washing the magnetic solid phase extraction material with a fourth solvent before eluting the target from the magnetic solid phase extraction material with a third solvent Phase extraction material. The purpose of the washing step is to remove impurities that may be non-specifically adsorbed to the magnetic SPE material before the target is eluted. It will be appreciated that, similar to the second solvent, the fourth solvent may be a poor solvent for the target. It can also be understood that, in order to avoid the loss of the target substance caused by the washing step, the solubility of the target substance in the fourth solvent used in the washing step should be much lower than that in the third solvent used in the elution step, for example, a maximum of only It is 50% of its solubility in the third solvent used for the elution step, preferably up to only 30%, more preferably up to only 10%. Most preferably, the target is completely insoluble in the fourth solvent used in the washing step.
如上所述，第一溶剂和第三溶剂均为目标物的良溶剂。因此，在又一具体的实施方案中，所述第一溶剂与所述第三溶剂是相同或不同的，可以各自独立地选自C
1-C
6一元醇，C
1-C
6多元醇，乙腈，乙酸乙酯，取代的甲烷，丙酮，苯或取代的苯，或其任意组合，但不限于此。
As described above, both the first solvent and the third solvent are good solvents for the target. Therefore, in yet another specific embodiment, the first solvent and the third solvent are the same or different, and can be independently selected from C 1 -C 6 monohydric alcohols, C 1 -C 6 polyhydric alcohols, Acetonitrile, ethyl acetate, substituted methane, acetone, benzene or substituted benzene, or any combination thereof, but not limited thereto.
在一个进一步具体的实施方案中，所述C
1-C
6一元醇可以为甲醇、乙醇或丙醇等，但不限于此。
In a further specific embodiment, the C 1 -C 6 monohydric alcohol may be methanol, ethanol or propanol, etc., but is not limited thereto.
在又一个进一步具体的实施方案中，所述C
1-C
6多元醇可以为乙二醇或丙二醇等，但不限于此。
In yet another further specific embodiment, the C 1 -C 6 polyol may be ethylene glycol or propylene glycol, etc., but is not limited thereto.
在又一个进一步具体的实施方案中，所述取代的甲烷可以为二氯甲烷、三氯甲烷、四氯甲烷等，但不限于此。In yet another further specific embodiment, the substituted methane may be dichloromethane, trichloromethane, tetrachloromethane, etc., but is not limited thereto.
在又另一个进一步具体的实施方案中，所述取代的苯可以为甲苯、二甲苯等，但不限于此。In yet another further specific embodiment, the substituted benzene may be toluene, xylene, etc., but is not limited thereto.
在一个任选的实施方案中，所述第一溶剂中掺杂有酸，例如有机酸或无机酸；或碱，例如有机碱或无机碱。In an optional embodiment, the first solvent is doped with an acid, such as an organic or inorganic acid; or a base, such as an organic or inorganic base.
在一个进一步具体的实施方案中，所述有机酸可以为三氟乙酸、三氯乙酸、甲酸、乙酸、丙酸、或对甲苯磺酸，但不限于此。In a further specific embodiment, the organic acid may be trifluoroacetic acid, trichloroacetic acid, formic acid, acetic acid, propionic acid, or p-toluenesulfonic acid, but is not limited thereto.
在一个进一步具体的实施方案中，所述无机酸可以为盐酸、硝酸、或硫酸，但不限于此。In a further specific embodiment, the inorganic acid may be hydrochloric acid, nitric acid, or sulfuric acid, but is not limited thereto.
在一个进一步具体的实施方案中，所述有机碱可以为三乙胺、三丙胺、或甲胺，但不限于此。In a further specific embodiment, the organic base may be triethylamine, tripropylamine, or methylamine, but is not limited thereto.
在一个进一步具体的实施方案中，所述无机碱可以为碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、氢氧化钠、或磷酸氢二钠，但不限于此。In a further specific embodiment, the inorganic base may be sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, or disodium hydrogen phosphate, but is not limited thereto.
如上所述，第二溶剂和第四溶剂均为目标物的不良溶剂。因此，在又一具体的实施方案中，所述第二溶剂与所述第四溶剂可以是相同或不同的，并且可以各自独立地选自液态烷烃，醚类，或其任意组合。As described above, both the second solvent and the fourth solvent are poor solvents for the target. Thus, in yet another specific embodiment, the second solvent and the fourth solvent may be the same or different, and may each be independently selected from liquid alkanes, ethers, or any combination thereof.
在一个进一步具体的实施方案中，所述液态烷烃可以为正己烷、环己烷等，但不限于此。In a further specific embodiment, the liquid alkane may be n-hexane, cyclohexane, etc., but is not limited thereto.
在一个进一步具体的实施方案中，所述醚类可以为石油醚、乙醚等，但不限于此。In a further specific embodiment, the ethers can be petroleum ether, diethyl ether, etc., but are not limited thereto.
在又一具体的实施方案中，所述磁性固相萃取材料为磁性纳米材料，例如但不限于磁性二氧化硅纳米颗粒、磁性高分子纳米颗粒、磁性富勒烯纳米颗粒等。In yet another specific embodiment, the magnetic solid phase extraction material is a magnetic nanomaterial, such as but not limited to magnetic silica nanoparticles, magnetic polymer nanoparticles, magnetic fullerene nanoparticles, and the like.
在又一具体的实施方案中，所述目标物为痕量目标物。In yet another specific embodiment, the target is a trace target.
在本发明的上下文中，术语“痕量”如本领域技术人员所理解的，指极少的量，例如含量低于百万分之一的量。由于其含量极低，固相萃取材料对于良溶剂中的痕量目标物的吸附也会更加困难，效率更低。而痕量目标物在不良溶剂中能够很好的分散，由此促进固相萃取材料对痕量目标物的吸附。因此，本发明的方法特别适用于痕量目标物的固相萃取。In the context of the present invention, the term "trace amount", as understood by a person skilled in the art, refers to a very small amount, eg an amount below one part per million. Due to its extremely low content, the adsorption of trace targets in good solvents by solid-phase extraction materials will also be more difficult and less efficient. The trace target can be well dispersed in the poor solvent, thereby promoting the adsorption of the trace target by the solid phase extraction material. Therefore, the method of the present invention is particularly suitable for solid phase extraction of trace targets.
在又一具体的实施方案中，所述目标物可以为小分子化合物、核酸、磷脂、以及蛋白质或多肽等。In yet another specific embodiment, the target can be a small molecule compound, nucleic acid, phospholipid, and protein or polypeptide.
在本发明的上下文中，术语“小分子化合物”是指分子量很小的化合物，通常是指分子量小于1000道尔顿、尤其是分子量小于500道尔顿的化合物。在一个具体的实施方案中，所述小分子化合物可以为农药残留，例如但不限于杀虫剂、杀菌剂、杀藻剂、除虫剂、落叶剂；兽药残留，例如但不限于抗生素类、磺胺类、呋喃类、抗寄生虫类、激素类药物等；真菌毒素，例如但不限于黄曲霉毒素；海洋毒素，例如但不限于河豚毒素、西加毒素等；非法食品添加剂，例如但不限于漂白剂、苏丹红、三聚氰胺等；重金属残留，例如但不限于汞、铅、镉等。In the context of the present invention, the term "small molecule compound" refers to a compound with a very small molecular weight, usually a compound with a molecular weight of less than 1000 Daltons, especially a molecular weight of less than 500 Daltons. In a specific embodiment, the small molecule compound can be pesticide residues, such as but not limited to pesticides, fungicides, algaecides, insecticides, defoliants; veterinary drug residues, such as but not limited to antibiotics, Sulfonamides, furans, antiparasitic drugs, hormone drugs, etc.; mycotoxins, such as but not limited to aflatoxins; marine toxins, such as but not limited to tetrodotoxin, cigatoxin, etc.; illegal food additives, such as but not limited to Bleach, Sudan red, melamine, etc.; heavy metal residues, such as but not limited to mercury, lead, cadmium, etc.
在又一具体的实施方案中，所述核酸可以为DNA或RNA。In yet another specific embodiment, the nucleic acid may be DNA or RNA.
在又一具体的实施方案中，所述蛋白质或多肽可以为酪蛋白、乳清蛋白、葡聚糖磷酸化酶、细胞色素C氧化酶、N-糖蛋白延胡索酸酶等，但不限于此。In yet another specific embodiment, the protein or polypeptide may be casein, whey protein, glucan phosphorylase, cytochrome C oxidase, N-glycoprotein fumarate, etc., but not limited thereto.
在又一具体的实施方案中，所述待测样本为食品或饲料。In yet another specific embodiment, the sample to be tested is food or feed.
在一个进一步具体的实施方案中，所述食品可以为乳制品，蔬菜如 马铃薯、番茄等，水果如甘蔗、苹果、梨等，水产品如鱼、虾、贝等，肉类如猪肉、牛肉、鸡肉等，粮油制品如小麦、面粉、黑米、菜籽油、大豆油等，保健食品如胶囊、粉剂等，但不限于此。In a further specific embodiment, the food can be dairy products, vegetables such as potatoes, tomatoes, etc., fruits such as sugar cane, apples, pears, etc., aquatic products such as fish, shrimp, shellfish, etc., meats such as pork, beef, Chicken, etc., grain and oil products such as wheat, flour, black rice, rapeseed oil, soybean oil, etc., health food such as capsules, powder, etc., but not limited to this.
在一个进一步具体的实施方案中，所述饲料可以为如家禽、水产等动物的饲料，但不限于此。In a further specific embodiment, the feed may be animal feed such as poultry, aquatic products, etc., but is not limited thereto.
在第二方面，本发明提供了一种待测样本中是否存在目标物的检测方法，所述方法包括实施第一方面所述的磁性固相萃取方法。In a second aspect, the present invention provides a method for detecting whether a target exists in a sample to be tested, the method comprising implementing the magnetic solid phase extraction method described in the first aspect.
可以理解，本发明第二方面包括上文针对本发明第一方面的方法进行描述的各特征和优点，在此不再赘述。It can be understood that the second aspect of the present invention includes the various features and advantages described above with respect to the method of the first aspect of the present invention, which will not be repeated here.
在一个具体的实施方案中，所述方法还包括进行免疫检测步骤或色谱分析步骤。In a specific embodiment, the method further comprises performing an immunodetection step or a chromatographic analysis step.
在一个具体的实施方案中，所述免疫检测可以为侧向流层析、酶联免疫、化学发光，但不限于此。In a specific embodiment, the immunodetection can be lateral flow chromatography, enzyme-linked immunosorbent assay, or chemiluminescence, but is not limited thereto.
在又一具体的实施方案中，所述色谱分析可以为高效液相色谱、液质联用、气质联用检测，但不限于此。In yet another specific embodiment, the chromatographic analysis can be high performance liquid chromatography, liquid chromatography-mass spectrometry, or gas-mass spectrometry detection, but is not limited thereto.
下文中，结合具体实施例更详细地描述本发明。然而，本文公开的具体实施例仅出于示例的目的，而不应该被认为旨在解释本发明的范围。Hereinafter, the present invention is described in more detail with reference to specific examples. However, the specific embodiments disclosed herein are for purposes of illustration only, and should not be considered as interpreting the scope of the invention.
实施例1：用于鱼中氯霉素的免疫检测Example 1: Immunodetection for Chloramphenicol in Fish
取24g鱼肉搅碎，平均分为4份，即样品1-4。分别向其中加入氯霉素标准溶液使得样品含氯霉素为0.2μg/L，加入6mL乙酸乙酯，涡旋3分钟。离心后取2mL上清液至5mL离心管。然后，在样品1和2中直接加入磁性二氧化硅纳米球，涡旋两分钟。对于样品3和4，首先经空气泵作用加热至60℃至溶剂挥干，然后加入1mL石油醚以及磁性二氧化硅纳米球，涡旋两分钟。利用磁铁将磁性二氧化硅纳米球吸附于管壁，然后将溶液移走。再移走磁铁，加入500μL 20％甲醇的Tris-HCl缓冲液(pH7.5，100mM)作为洗脱液，涡旋1分钟。再次利用磁铁将 磁性二氧化硅纳米球吸附于管壁，吸取洗脱液200μL，将其加入包含偶联了氯霉素抗体的胶体金容器中，于35℃反应3分钟。加入氯霉素试纸条，继续反应3分钟，然后通过比色法(即，免疫层析上T/C线色度比值)进行结果判读，结果列于下表1。Take 24g of fish meat and chop it up and divide it into 4 parts, namely samples 1-4. A standard solution of chloramphenicol was added to each to make the sample containing chloramphenicol at 0.2 μg/L, 6 mL of ethyl acetate was added, and vortexed for 3 minutes. After centrifugation, 2 mL of supernatant was transferred to a 5 mL centrifuge tube. Then, magnetic silica nanospheres were added directly to samples 1 and 2 and vortexed for two minutes. For samples 3 and 4, they were first heated to 60 °C by an air pump to evaporate the solvent, then 1 mL of petroleum ether and magnetic silica nanospheres were added, and vortexed for two minutes. The magnetic silica nanospheres were adsorbed to the tube wall with a magnet, and then the solution was removed. The magnet was removed again, 500 μL of 20% methanol in Tris-HCl buffer (pH 7.5, 100 mM) was added as eluent, and vortexed for 1 minute. The magnetic silica nanospheres were again adsorbed on the tube wall with a magnet, and 200 μL of the eluate was drawn, added to the colloidal gold container containing the conjugated chloramphenicol antibody, and reacted at 35°C for 3 minutes. Chloramphenicol test strips were added, and the reaction was continued for 3 minutes, and then the results were judged by colorimetry (ie, T/C line chromaticity ratio on immunochromatography). The results are listed in Table 1 below.
表1：鱼中氯霉素的胶体金检测结果Table 1: Colloidal gold detection results for chloramphenicol in fish
当T/C(即表中读数)＜1为阳性；＞1为阴性；＝1为需要复测。由表1可知，直接加入磁性二氧化硅纳米球的样品1和2，经胶体金检测结果显示均为阴性；而对于经挥干然后加入石油醚处理的样品3和4，则经胶体金检测结果显示均为阳性。该结果表明，相比直接的磁性固相萃取方法，本发明的经溶剂置换的磁性固相萃取方法能够更灵敏、高效、准确地检测低浓度的小分子残留如氯霉素，由此更适用于动植物如水产中的农药残留的检测。When T/C (that is, the reading in the table) < 1, it is positive; > 1 is negative; = 1, retest is required. As can be seen from Table 1, the samples 1 and 2 that were directly added with magnetic silica nanospheres were negatively tested by colloidal gold; while for samples 3 and 4, which were evaporated and then added with petroleum ether, they were detected by colloidal gold. The results were all positive. The results show that, compared with the direct magnetic solid-phase extraction method, the magnetic solid-phase extraction method by solvent replacement of the present invention can detect low-concentration small molecule residues such as chloramphenicol more sensitively, efficiently and accurately, and thus is more applicable For the detection of pesticide residues in animals and plants such as aquatic products.
实施例2：用于玉米中黄曲霉毒素B1的HPLC检测Example 2: HPLC detection of aflatoxin B1 in corn
取20g玉米粒搅碎，加入24毫升乙腈，涡旋3分钟。离心后分别取2mL上清液加入4个离心管，即样品1-4。然后，向其中加入黄曲霉毒素B1标准溶液使得终浓度为10μg/L。其中，向样品1和2中直接加入磁性二氧化硅纳米球，涡旋2分钟。对于样品3和4，首先经空气泵作用加热至75℃至其中溶剂挥干，然后加入1mL正己烷以及磁性二氧化硅纳米球，涡旋2分钟。利用磁铁将磁性二氧化硅纳米球吸附于管壁，然后移取溶液。利用0.22μm滤膜过滤该溶液，然后通过LCMS/MS进行测量，并计算经该溶液中黄曲霉毒素B1的浓度，由此计算磁性二氧化硅纳米球在样品1-4中对黄曲霉毒素B1的吸附率，结果列于下表2。 表2：经吸附后的溶液中黄曲霉毒素B1浓度和磁性二氧化硅纳米球的吸附率20 g of corn kernels were crushed, 24 ml of acetonitrile was added, and vortexed for 3 minutes. After centrifugation, 2 mL of supernatant was added to 4 centrifuge tubes, namely samples 1-4. Then, a standard solution of aflatoxin B1 was added thereto so that the final concentration was 10 μg/L. Among them, magnetic silica nanospheres were directly added to samples 1 and 2 and vortexed for 2 minutes. For samples 3 and 4, they were first heated to 75° C. by an air pump to evaporate the solvent, then 1 mL of n-hexane and magnetic silica nanospheres were added, and vortexed for 2 minutes. The magnetic silica nanospheres were adsorbed on the tube wall with a magnet, and then the solution was pipetted. The solution was filtered with a 0.22 μm filter, then measured by LCMS/MS, and the concentration of aflatoxin B1 in the solution was calculated, thereby calculating the effect of magnetic silica nanospheres on aflatoxin B1 in samples 1-4. The adsorption rate of , the results are listed in Table 2 below. Table 2: The concentration of aflatoxin B1 in the adsorbed solution and the adsorption rate of magnetic silica nanospheres
| 样品sample | 黄曲霉毒素B1浓度(μg/L)Aflatoxin B1 concentration (μg/L) | 吸附率Adsorption rate |
| 11 | 7.777.77 | 22.3％22.3% |
| 22 | 8.108.10 | 19.0％19.0% |
| 33 | 0.850.85 | 91.5％91.5% |
| 44 | 1.011.01 | 89.9％89.9% |
由上表2可知，相比直接的磁性固相萃取方法，根据本发明的经溶剂置换的磁性固相萃取方法能够更高效地吸附目标物，吸附率由样品1-2(直接法)的19.0％-22.3％提高至样品1-4的89.9％-91.5％，吸附率是直接法的约4-6倍。It can be seen from the above table 2 that compared with the direct magnetic solid phase extraction method, the magnetic solid phase extraction method by solvent replacement according to the present invention can adsorb the target substance more efficiently, and the adsorption rate is 19.0% higher than that of sample 1-2 (direct method). %-22.3% increased to 89.9%-91.5% of samples 1-4, and the adsorption rate was about 4-6 times that of the direct method.
Claims (15)
- 一种磁性固相萃取方法，其包括：A magnetic solid-phase extraction method, comprising:a)使用第一溶剂从待测样本制备含有目标物和所述第一溶剂的第一溶液；a) using a first solvent to prepare a first solution containing the target and the first solvent from the sample to be tested;b)加入第二溶剂以置换所述第一溶剂，从而获得第二溶液，其中，所述目标物在所述第二溶剂中的溶解度低于在所述第一溶剂中的溶解度；b) adding a second solvent to replace the first solvent, thereby obtaining a second solution, wherein the solubility of the target in the second solvent is lower than the solubility in the first solvent;c)向步骤b)中得到的第二溶液中加入磁性固相萃取材料以吸附所述目标物。c) adding a magnetic solid phase extraction material to the second solution obtained in step b) to adsorb the target substance.
- 根据权利要求1所述的方法，其中，所述方法还包括：在进行步骤b)之前，先例如通过热、和/或气流、和/或真空使所述第一溶剂挥干的步骤。The method according to claim 1, wherein the method further comprises the step of volatilizing the first solvent, for example by heat, and/or gas flow, and/or vacuum, before performing step b).
- 根据权利要求1或2中任一项所述的方法，其中，所述第二溶剂的体积与所述第一溶剂的体积相同或不同，优选地所述第二溶剂的体积少于所述第一溶剂的体积。The method according to any one of claims 1 or 2, wherein the volume of the second solvent is the same as or different from the volume of the first solvent, preferably the volume of the second solvent is less than the volume of the first solvent a volume of solvent.
- 根据权利要求1-3中任一项所述的方法，其中，在所述第二溶剂中所述磁性固相萃取材料对所述目标物的吸附率为至少50％，优选为至少70％，更优选为至少90％，甚至更优选为至少99％。The method according to any one of claims 1-3, wherein the adsorption rate of the target substance by the magnetic solid phase extraction material in the second solvent is at least 50%, preferably at least 70%, More preferably at least 90%, even more preferably at least 99%.
- 根据权利要求1-4中任一项所述的方法，其还包括：在步骤c)之后，先分离所述第二溶液与所述磁性固相萃取材料，然后用第三溶剂从所述磁性固相萃取材料中洗脱所述目标物，以获得含有所述目标物的第三溶液，其中，所述目标物在所述第三溶剂中的溶解度高于在所述第二溶剂中的溶解度。The method according to any one of claims 1-4, further comprising: after step c), first separating the second solution and the magnetic solid phase extraction material, and then using a third solvent to remove the magnetic solid phase extraction material from the magnetic The target substance is eluted from the solid phase extraction material to obtain a third solution containing the target substance, wherein the solubility of the target substance in the third solvent is higher than that in the second solvent .
- 根据权利要求1-5中任一项所述的方法，其中，在所述第二溶剂中所述磁性固相萃取材料对所述目标物的吸附率高于在所述第一溶剂和/或所述第三溶剂中所述磁性材料对所述目标物的吸附率。The method according to any one of claims 1-5, wherein the adsorption rate of the magnetic solid phase extraction material to the target in the second solvent is higher than that in the first solvent and/or The adsorption rate of the magnetic material to the target in the third solvent.
- 根据权利要求5-6中任一项所述的方法，其还包括：在用第 三溶剂从所述磁性固相萃取材料中洗脱所述目标物之前，使用第四溶剂洗涤所述磁性固相萃取材料。The method of any one of claims 5-6, further comprising: washing the magnetic solid phase extraction material with a fourth solvent before eluting the target from the magnetic solid phase extraction material with a third solvent Phase extraction material.
- 根据权利要求1-7中任一项所述的方法，其中，所述第一溶剂与所述第三溶剂是相同或不同的，各自独立地选自C 1-C 6一元醇如甲醇、乙醇、丙醇，C 1-C 6多元醇如乙二醇、丙二醇，乙腈，乙酸乙酯，取代的甲烷如二氯甲烷、三氯甲烷、四氯甲烷，丙酮，苯或取代的苯如甲苯、二甲苯，或其任意组合；并且任选地所述第一溶剂掺杂有酸如有机酸或无机酸，或碱如有机碱或无机碱；具体地，所述有机酸例如为三氟乙酸、三氯乙酸、甲酸、乙酸、丙酸、或对甲苯磺酸；所述无机酸例如为盐酸、硝酸、或硫酸；所述有机碱例如为三乙胺、三丙胺、或甲胺；所述无机碱例如为碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、氢氧化钠、或磷酸氢二钠。 The method according to any one of claims 1-7, wherein the first solvent and the third solvent are the same or different, each independently selected from C 1 -C 6 monohydric alcohols such as methanol, ethanol , Propanol, C 1 -C 6 polyols such as ethylene glycol, propylene glycol, acetonitrile, ethyl acetate, substituted methanes such as dichloromethane, chloroform, tetrachloromethane, acetone, benzene or substituted benzenes such as toluene, Xylene, or any combination thereof; and optionally the first solvent is doped with an acid such as an organic acid or an inorganic acid, or a base such as an organic base or an inorganic base; specifically, the organic acid is, for example, trifluoroacetic acid, Trichloroacetic acid, formic acid, acetic acid, propionic acid, or p-toluenesulfonic acid; the inorganic acid is, for example, hydrochloric acid, nitric acid, or sulfuric acid; the organic base is, for example, triethylamine, tripropylamine, or methylamine; The base is, for example, sodium carbonate, potassium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, sodium hydroxide, or disodium hydrogenphosphate.
- 根据权利要求1-8中任一项所述的方法，其中，所述第二溶剂与所述第四溶剂是相同或不同的，各自独立地选自液态烷烃例如正己烷、环己烷，醚类例如石油醚、乙醚、或其任意组合。The method according to any one of claims 1-8, wherein the second solvent and the fourth solvent are the same or different, each independently selected from liquid alkanes such as n-hexane, cyclohexane, ethers such as petroleum ether, diethyl ether, or any combination thereof.
- 根据权利要求1-9中任一项所述的方法，其中，所述磁性固相萃取材料为磁性纳米材料如磁性二氧化硅纳米颗粒、磁性高分子纳米颗粒、磁性富勒烯纳米颗粒、磁性碳纳米管颗粒。The method according to any one of claims 1-9, wherein the magnetic solid phase extraction material is a magnetic nanomaterial such as magnetic silica nanoparticles, magnetic polymer nanoparticles, magnetic fullerene nanoparticles, magnetic Carbon Nanotube Particles.
- 根据权利要求1-10中任一项所述的方法，其中，所述目标物为痕量目标物。The method of any one of claims 1-10, wherein the target is a trace target.
- 根据权利要求1-11中任一项所述的方法，其中，所述目标物为小分子化合物如农药残留、兽药残留、真菌毒素、海洋毒素、非法食品添加剂、重金属残留，核酸如DNA、RNA，磷脂，以及蛋白质或多肽如酪蛋白、乳清蛋白、葡聚糖磷酸化酶、细胞色素C氧化酶、N-糖蛋白延胡索酸酶等。The method according to any one of claims 1-11, wherein the target is small molecule compounds such as pesticide residues, veterinary drug residues, mycotoxins, marine toxins, illegal food additives, heavy metal residues, nucleic acids such as DNA, RNA , phospholipids, and proteins or polypeptides such as casein, whey protein, glucan phosphorylase, cytochrome C oxidase, N-glycoprotein fumarate, etc.
- 根据权利要求1-12中任一项所述的方法，其中，所述待测样本为食品，例如乳制品，蔬菜如马铃薯、番茄，水果如甘蔗、苹果、梨，水产品如鱼、虾、贝，肉类如猪肉、牛肉、鸡肉，粮油制品如小麦、面粉、黑米、菜籽油、大豆油，保健食品如胶囊、粉剂， 或饲料。The method according to any one of claims 1-12, wherein the sample to be tested is food, such as dairy products, vegetables such as potatoes, tomatoes, fruits such as sugar cane, apples, pears, aquatic products such as fish, shrimp, Shellfish, meat such as pork, beef, chicken, grain and oil products such as wheat, flour, black rice, rapeseed oil, soybean oil, health food such as capsules, powders, or feed.
- 一种待测样本中是否存在目标物的检测方法，所述方法包括实施权利要求1-13中任一项所述的磁性固相萃取方法。A method for detecting whether a target substance exists in a sample to be tested, the method comprising implementing the magnetic solid phase extraction method according to any one of claims 1-13.
- 根据权利要求14所述的方法，所述方法还包括进行免疫检测如侧向流层析、酶联免疫、化学发光的步骤，或进行色谱分析如高效液相色谱、液质联用、气质联用的步骤。The method according to claim 14, further comprising the step of performing immunodetection such as lateral flow chromatography, ELISA, chemiluminescence, or performing chromatographic analysis such as high performance liquid chromatography, LC-MS, GC-MS steps used.
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