CN116068106A - Affinity monolithic column of hydrophobic matrix ultrahigh-density modified aptamer and preparation method thereof - Google Patents
Affinity monolithic column of hydrophobic matrix ultrahigh-density modified aptamer and preparation method thereof Download PDFInfo
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 51
- 239000011159 matrix material Substances 0.000 title claims abstract description 47
- 230000002209 hydrophobic effect Effects 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000000178 monomer Substances 0.000 claims abstract description 34
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 19
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims abstract description 16
- 150000001768 cations Chemical class 0.000 claims abstract description 14
- 229920000734 polysilsesquioxane polymer Polymers 0.000 claims abstract description 8
- 238000004132 cross linking Methods 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 22
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 claims description 14
- 239000002202 Polyethylene glycol Substances 0.000 claims description 14
- ISAOCJYIOMOJEB-UHFFFAOYSA-N benzoin Chemical group C=1C=CC=CC=1C(O)C(=O)C1=CC=CC=C1 ISAOCJYIOMOJEB-UHFFFAOYSA-N 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- 108091008104 nucleic acid aptamers Proteins 0.000 claims description 14
- 229920001223 polyethylene glycol Polymers 0.000 claims description 14
- 239000003999 initiator Substances 0.000 claims description 13
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 12
- PSGAAPLEWMOORI-PEINSRQWSA-N medroxyprogesterone acetate Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2CC[C@]2(C)[C@@](OC(C)=O)(C(C)=O)CC[C@H]21 PSGAAPLEWMOORI-PEINSRQWSA-N 0.000 claims description 12
- 239000010453 quartz Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical group FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 11
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 11
- 238000006116 polymerization reaction Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 125000003342 alkenyl group Chemical group 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 244000028419 Styrax benzoin Species 0.000 claims description 7
- 235000000126 Styrax benzoin Nutrition 0.000 claims description 7
- 235000008411 Sumatra benzointree Nutrition 0.000 claims description 7
- 229960002130 benzoin Drugs 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 235000019382 gum benzoic Nutrition 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- -1 acrylic ester Chemical class 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000007872 degassing Methods 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- RRHXZLALVWBDKH-UHFFFAOYSA-M trimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azanium;chloride Chemical group [Cl-].CC(=C)C(=O)OCC[N+](C)(C)C RRHXZLALVWBDKH-UHFFFAOYSA-M 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 125000005397 methacrylic acid ester group Chemical group 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 150000007523 nucleic acids Chemical group 0.000 claims description 2
- 230000000379 polymerizing effect Effects 0.000 claims description 2
- UJKWLAZYSLJTKA-UHFFFAOYSA-N edma Chemical compound O1CCOC2=CC(CC(C)NC)=CC=C21 UJKWLAZYSLJTKA-UHFFFAOYSA-N 0.000 claims 5
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 229930183344 ochratoxin Natural products 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 6
- 238000000707 layer-by-layer assembly Methods 0.000 abstract description 3
- 229920000447 polyanionic polymer Polymers 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000010526 radical polymerization reaction Methods 0.000 abstract description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 abstract 1
- 230000005526 G1 to G0 transition Effects 0.000 abstract 1
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 description 19
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 description 18
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 description 17
- 238000001179 sorption measurement Methods 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- JHPBZFOKBAGZBL-UHFFFAOYSA-N (3-hydroxy-2,2,4-trimethylpentyl) 2-methylprop-2-enoate Chemical group CC(C)C(O)C(C)(C)COC(=O)C(C)=C JHPBZFOKBAGZBL-UHFFFAOYSA-N 0.000 description 1
- MTEZSDOQASFMDI-UHFFFAOYSA-N 1-trimethoxysilylpropan-1-ol Chemical compound CCC(O)[Si](OC)(OC)OC MTEZSDOQASFMDI-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
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- 238000000861 blow drying Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 description 1
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- 239000002636 mycotoxin Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
- G01N30/6073—Construction of the column body in open tubular form
- G01N30/6078—Capillaries
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids
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Abstract
The invention belongs to the field of analytical chemistry, and particularly relates to an affinity monolithic column of a hydrophobic matrix ultra-high density modified aptamer and a preparation method thereof. The affinity monolithic column takes quaternary ammonium salt strong cations and 2-oxazoline-5-ketone reagents as monomers, acrylate monomers or polysilsesquioxane as cross-linking agents, and forms a strong cation matrix stationary phase through photoinitiated free radical polymerization reaction; based on the fact that the aptamer is polyanion, the aptamer affinity monolithic column with ultrahigh coverage density is prepared by fixing the aptamer to the surface of a strong cation monolithic column matrix through electrostatic self-assembly. The monolithic column matrix has a high-crosslinking structure and strong charge effect, and can realize the direct high-density load fixation of the aptamer of the natural base on the monolithic column.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to an affinity monolithic column of a hydrophobic matrix ultra-high density modified aptamer and a preparation method thereof.
Background
The aptamer serving as an emerging material has the characteristics of high affinity, good stability, in-vitro screening and synthesis, wide target ligand range and the like, and is widely applied to the selective extraction, separation and detection of target analytes in complex samples. The existing developed polymerization monolithic column generally adopts methacrylate monomers, and the prepared monolithic column contains hydrophobic organic polymerization monomers or cross-linking agents and has the advantages of good preparation reaction stability, low swelling in aqueous phase polar environment, high commercialization degree of monomers, easy obtainment and the like. However, as the conventional monomers such as EDMA, POSS and the like have strong hydrophobicity, the prepared affinity monolithic column has strong hydrophobicity, and the hydrophobic matrix has serious nonspecific adsorption effect on the organic matters such as hydrophobic mycotoxins and the like. How to develop a new technology, eliminate the nonspecific effect of the hydrophobic matrix affinity monolithic column, ensure the specific recognition capability of the hydrophobic matrix monolithic column, and have good research significance for popularizing the high-efficiency affinity recognition application of the affinity monolithic column.
At present, two main technical modes exist, one is to improve the hydrophilicity of the monolithic column matrix and inhibit the adsorption of hydrophobic coexisting materials based on the hydrophilicity, but the characteristic of the polymeric matrix needs to be changed; the other way improves the coverage density of the aptamer on the surface of the hydrophobic matrix, and directly masks the hydrophobic adsorption sites of the matrix, thereby avoiding nonspecific adsorption. The development of functionalized affinity monolithic columns with high coverage density of aptamer becomes an effective strategy for improving specific recognition capability of organic polymeric monolithic columns. Yu et al prepared POSS-based monolithic columns by thermally initiated polymerization, followed by subsequent aptamer binding by AuNPs or cyanuric chloride (TCT) with a coverage density of up to 1413-1799 pmol. Mu.L -1 However, due to insufficient aptamer capacity and hydrophobic interaction of the matrix, nonspecific adsorption is still obvious (recovery rate of analogues is 6.5-8.3%). Xu and the like have high coverage density of 2772.61 pmol/mu L through thiol-alkenyl click chemical grafting aptamer, multiple steps are needed, the time is as long as 36-52 hours, the adopted aptamer needs to be introduced with special sulfhydryl groups and the like, the reagent price is high, the problem of nonspecific adsorption still exists, and the recovery rate of the analogue is 3.27%. Surface high density covering coreThe preparation efficiency and economy of the hydrophobic affinity monolithic column of the acid aptamer are still at a low level, and the solution is needed.
The research of the invention shows that the aptamer is formed by assembling nucleotide bases, is not only an active site specifically recognized, but also has strong polarity (charge) per se. Through electrostatic action, the cationic groups can effectively adsorb the anionic aptamer, a high-coverage-density aptamer modification layer is directly formed on the surface of the cationic polymer, more recognition sites can be provided on the monolithic column, the non-specific adsorption effect is reduced to the greatest extent, the adopted method is electrostatic adsorption, no special group modification is needed, the method is simple and efficient, and no related report has been made yet.
Disclosure of Invention
The invention aims to provide an affinity monolithic column of a hydrophobic matrix ultrahigh-density modified aptamer and a preparation method thereof. The invention introduces strong cation monomer and rapidly prepares strong cation monolithic column matrix based on photoinitiated polymerization technology. A large number of quaternary ammonium groups on the surface of the matrix column can provide stable positive charges, and the polyanion aptamer with a phosphate skeleton can be rapidly fixed on the matrix column through the electrostatic self-assembly effect, so that the aptamer affinity monolithic column with ultrahigh coverage density is obtained.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the affinity monolithic column is formed by polymerizing a quaternary ammonium salt strong cation functional monomer, a 2-oxazoline-5-ketone monomer and an acrylic ester cross-linking agent or a polysilsesquioxane cross-linking agent under the assistance of a pore-forming agent and an initiator by ultraviolet light to form a hydrophobic monolithic column matrix with the surface rich in positive charges, and then self-assembling and adsorbing a nucleic acid aptamer sequence consisting of natural bases based on electrostatic action to form the affinity monolithic column with the surface functionalized hydrophobic matrix ultrahigh density aptamer.
Further, the quaternary ammonium salt strong cation functional monomer is methacryloxyethyl trimethyl ammonium chloride (DMC), the 2-oxazoline-5-ketone monomer is 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone (VDMA), the acrylic ester cross-linking agent is ethylene glycol dimethacrylate (EDMA), and the polysilsesquioxane cross-linking agent is methacrylic ester grafted cage-type oligomeric silsesquioxane (POSS-MA).
Further, the pore-forming agent is a binary pore-forming agent consisting of N, N-Dimethylformamide (DMF) and polyethylene glycol (PEG-10000) with molecular weight of 10000; the initiator is benzoin dimethyl ether (DMPA).
Further, the total mass percentage of the components is 100 percent, the DMC of the strong cationic functional monomer of quaternary ammonium salt is 9.36 to 10.25 percent, the EDMA of the acrylic ester cross-linking agent is 7.28 to 7.97 percent, the VDMA of the 2-oxazoline-5-ketone monomer is 4.16 to 4.55 percent, the DMF of N, N-dimethylformamide is 67.96 to 71.94 percent, the PEG-10000 of polyethylene glycol is 6.10 to 9.26 percent, and the DMPA of the initiator benzoin dimethyl ether is 1.0 percent; or the quaternary ammonium salt strong cation functional monomer DMC is 7.52-8.32%, polysilsesquioxane cross-linking agent POSS-MA is 6.93-8.91%, 2-oxazoline-5-ketone monomer VDMA is 4.70-5.20%, N-dimethylformamide DMF is 71.95-73.77%, polyethylene glycol PEG-10000 is 6.26-6.42%, and initiator benzoin dimethyl ether DMPA is 1.0%.
Further, the nucleic acid aptamer is a natural base composition nucleic acid sequence GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA of anti-Ochratoxin (OTA).
The preparation method of the hydrophilic monolithic column of the hydrophobic matrix ultra-high density modified aptamer comprises the following steps:
(1) Activation of anti-OTA toxin nucleic acid aptamer:
centrifuging the nucleic acid aptamer with the natural base composition for resisting OTA toxin at 6000 r/min for 15 min, adding 128 mu L of Tris-HCl buffer solution to dilute to 100 mu mol/L, carrying out vortex oscillation for 3 min, heating at 90 ℃ for 3 min, annealing, and cooling to room temperature for later use;
(2) Preparation of a polymerized monolithic column based on EDMA crosslinking:
weighing methacryloxyethyl trimethyl ammonium chloride DMC monomer, 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone VDMA monomer and ethylene glycol dimethacrylate EDMA cross-linking agent according to a proportion, adding pore-forming agent DMF and PEG-10000 and initiator DMPA, vortex oscillating for 3 min at room temperature, and performing ultrasonic degassing for 10 min to form a uniform solution; injecting the mixture into an alkenyl pretreated quartz capillary, sealing two ends, and placing the quartz capillary in 365 nm ultraviolet irradiation for 7 min for polymerization; after the reaction is completed, the prepared monolithic column is taken out and connected to a high-pressure solvent pump for a liquid chromatograph, and residues are removed by using methanol to obtain an EDMA cross-linked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges, which is hereinafter referred to as an EDV monolithic column;
(3) Preparation of a polymerized monolithic column based on POSS-MA crosslinking:
weighing methacryloxyethyl trimethyl ammonium chloride DMC monomer, 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone VDMA monomer and methacrylic acid ester grafted cage-type oligomeric silsesquioxane POSS-MA cross-linking agent according to a proportion, adding pore-forming agent DMF, PEG-10000 and initiator DMPA, carrying out vortex oscillation for 3 min at room temperature, and carrying out ultrasonic degassing for 10 min to form uniform solution; injecting the mixture into an alkenyl pretreated quartz capillary, sealing two ends, and placing the quartz capillary in 365 nm ultraviolet irradiation for 5 min for polymerization; after the reaction is completed, the prepared monolithic column is taken out and connected to a high-pressure solvent pump for a liquid chromatograph, and residues are removed by using methanol to obtain a POSS-MA cross-linked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges, which is hereinafter called PDV monolithic column;
(4) Preparation of an affinity monolithic column of a hydrophobic matrix ultra-high density modified aptamer:
filling the EDV monolithic column prepared in the step (2) or the PDV monolithic column prepared in the step (3) with Tris-HCl buffer solution, then injecting 20 mu L of the nucleic acid aptamer stock solution obtained in the step (1) through a liquid phase pump to prepare an affinity monolithic column of the ultra-high density modified aptamer, and storing at the temperature of 4 ℃.
Further, the Tris-HCl buffer pH was 8.00, consisting of 10mmol/L Tris-HCl,120 mmol/L NaCl and 5 mmol/L KCl.
The invention has the remarkable advantages that:
the invention provides an affinity monolithic column utilizing a hydrophobic matrix ultra-high density modified aptamer and a preparation method thereof. Based on photoinitiated free radical polymerization, a strong cationic monomer is introduced to rapidly prepare a monolithic column matrix with a large number of quaternary ammonium groups on the surface. Then, by utilizing the characteristic that the aptamer is linear polyanion, the ultra-high density coverage of the aptamer is easily realized through electrostatic self-assembly, the coverage density of the aptamer reaches 8616 (POSS crosslinking type) pmol/mu L and 6192 (acrylic ester crosslinking type) pmol/mu L respectively, and no modification is needed. The ultra-high coverage density effectively inhibits the problem of non-specific adsorption caused by the hydrophobic matrix. Compared with the traditional covalent bond bonding method, the method has the advantages of simplicity and rapidness in preparation, high immobilization efficiency of the aptamer, and the like. The affinity entity prepared by the invention has ultrahigh coverage density, can effectively reduce nonspecific adsorption, takes OTA as an analysis object, and has the recovery rate of only 0.55% when the concentration of the analogue is up to 250 times.
Drawings
FIG. 1 is an electron microscope image (A, C) and an enlarged image (B, D) of an aptamer affinity monolith based on EDV and PDV, respectively;
FIG. 2 is a comparison of the recognition of ochratoxin A by different monoliths, wherein (I) is a blank column of unmodified aptamer, (II) is a control column modified with aptamer without ochratoxin A, and (III) is an affinity monolith column modified with aptamer of ochratoxin A.
FIG. 3 is a graph showing the comparison of ochratoxin A and its analogues in different columns, wherein (A1, A2) are EDV and PDV matrix blank columns of unmodified nucleic acid aptamer, respectively, (B1, B2) are control columns of EDV and PDV matrix modified nucleic acid aptamer without ochratoxin A, respectively, and (C1, C2) are affinity columns of EDV and PDV matrix modified nucleic acid aptamer, respectively.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
A preparation method of an affinity monolithic column of a hydrophobic matrix ultra-high density modified aptamer comprises the following specific steps:
(1) Alkenyl derivative for inner wall of capillary column
Sequentially introducing 0.1 mol/L hydrochloric acid into a quartz capillary hollow column for 30 min, introducing secondary water to neutrality, introducing 0.1 mol/L sodium hydroxide solution 3h, then introducing secondary water again to neutrality, introducing methanol for 30 min, and blow-drying with nitrogen at 25 ℃ and 0.5 MPa; injecting a mixture of methanol and gamma-methacrylic acid oxypropyl trimethoxy silane (1:1, v/v) into the capillary tube, and reacting in a constant temperature water bath at 60 ℃ for 24 h to bond a silanized substance with alkenyl on the wall of the capillary tube; then the mixture is washed clean by methanol, and nitrogen is blown for 3 hours under the condition of 70 ℃ and 0.5MPa, thus obtaining the capillary column with the surface alkenyl (namely the quartz capillary with the alkenyl pretreated).
(2) Preparation of EDV polymerization monolithic column
The following table 1 shows that the total mass percentage of the components is 100%, DMC is 9.36-10.25%, EDMA is 7.28-7.97%, VDMA is 4.16-4.55%, DMF is 67.96-71.94%, PEG-10000 is 6.10-9.26%, DMPA is 1.0%, then it is added into centrifuge tube, vortex oscillating is carried out for 3 min at room temperature, ultrasonic degassing is carried out for 10 min, and uniform solution is formed; injecting the polymerization solution into the pretreated transparent alkenyl capillary tube, sealing two ends, and reacting for 7 min under the irradiation of 365 and nm ultraviolet light; taking out the prepared monolithic column, connecting to a high-pressure solvent pump for a liquid chromatograph, flushing the monolithic column by taking methanol as a mobile phase, and removing residues which are reacted to obtain the polymeric monolithic column with the surface containing quaternary ammonium salt positive charges, namely the EDV monolithic column.
Table 1 formulation (mass%) of EDV monolithic column
(3) Preparation of PDV polymerization monolithic column
The following Table 2 shows that the total mass percent of the components is 100%, the DMC of the quaternary ammonium salt strong cation functional monomer is 6.93-8.91%, the POSS-MA of the polysilsesquioxane crosslinking agent is 5.94-7.92%, the VDMA of the 2-oxazoline-5-ketone monomer is 4.70-5.20%, the DMF of N, N-dimethylformamide is 71.95-73.77%, the PEG-10000 of polyethylene glycol is 6.26-6.42%, the DMPA of the initiator benzoin dimethyl ether is 1.0%, vortex oscillation is carried out for 3 min at room temperature, and ultrasonic degassing is carried out for 20 min to form a uniform solution; injecting the mixture into an alkenyl pretreated quartz capillary, sealing two ends, and placing the quartz capillary in 365 nm ultraviolet irradiation for 5 min for polymerization; after the reaction is completed, the prepared monolithic column is taken out and connected to a high-pressure solvent pump for a liquid chromatograph, and residues are removed by using methanol to obtain the POSS-MA crosslinked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges, which is hereinafter referred to as PDV monolithic column.
Table 2 formulation of PEV monolith (mass%)
(4) Preparation of an affinity monolithic column of a hydrophobic matrix ultra-high density modified aptamer:
filling the EDV prepared in the step (2) or the PDV monolithic column prepared in the step (3) with Tris-HCl buffer solution, then injecting 20 mu L of the nucleic acid aptamer stock solution obtained in the step (1) through a liquid phase pump to prepare an affinity monolithic column of the ultra-high density modified aptamer, namely Apt@EDV and Apt@PDV affinity monolithic columns respectively, and storing at 4 ℃.
FIG. 1 shows the structure of the ultra-high density aptamer-covered affinity monolithic column of EDV matrix or PDV matrix, and the structure of the prepared aptamer-covered affinity monolithic column is complete and uniform, the filler is tightly combined with the column wall, and the column structure is stable.
Application example 1
And taking ochratoxin A (OTA) as an application object, and evaluating the specific recognition effect of the prepared affinity column on a target object. The whole column has a length of 5 cm, and is respectively balanced, loaded, washed and eluted, and the specific steps are as follows:
(1) Balance: the mixture is equilibrated with a binding buffer at a flow rate of 0.10. 0.10 mL/min and a pressure of 250psi for 0.5 hours, wherein the binding buffer is 10mmol/L Tris-HCl,120 mmol/L NaCl,5 mmol/L KCl and 20mmol/L CaCl 2 ，pH 8.00；
(2) Loading: injecting 20 mu L of sample solution (5 ng/mL) into the quantitative ring, taking the column No. 2 in the example as an application example, injecting the sample solution into the whole column No. 2 by a chromatographic pump, loading the sample solution for 0.5 hour, wherein the condition of the chromatographic pump is 0.1mL/min, and the pressure is 250psi; for experimental comparison, the same loading operation is adopted for the blank column and the comparison column respectively;
(3) Cleaning: loading the loaded aptamer modified monolithic column onto a liquid chromatographic pump, and cleaning the blank column, the control column and the aptamer affinity monolithic column by adopting a binding buffer solution;
(4) Eluting: with 30% acetonitrile ACN: eluting OTA from the monolithic column by using 70% TE buffer (10 mM Tris-HCl pH8.00,2.5mM EDTA) as eluent, and collecting the eluent at a flow rate of 0.1mL/min by using a 250psi back pressure valve to be detected; the same elution operation is also adopted for the blank column and the control column;
(5) And (3) detection: injecting the cleaning solution to be detected and the eluent into the HPLC-RF-20A for detection, and detecting OTA conditions: mobile phase: acetic acid aqueous solution (2%): acetonitrile=38:62, e x =333 nm，E m Results of 20 μl samples taken at 460 nm,1.0 mL/min are shown in fig. 2.
Bound to the control column is ssDNA randomization sequence CTG GCCCAG ATT TTA AGG TGCGTA AAG AAA AAA AGT.
As can be seen from fig. 2, in the loading process, more OTA was detected in the effluent from the ends of the blank column (EDV matrix column or PDV matrix column) and the control column and the washing liquid of the column, whereas for the affinity monolithic column, the loading flow effluent and washing liquid failed to detect significant OTA; for eluent, blank columns (EDV matrix columns or PDV matrix columns) and control columns do not effectively retain ochratoxin A, and OTA in the affinity monolithic column is effectively eluted, so that the aptamer affinity monolithic column prepared by the method can realize selective identification of ochratoxin A.
As can be seen from fig. 3, for the aptamer functionalized apt@edv and apt@pdv affinity monoliths, the OTA recovery was high in the eluate (C1, C2 in fig. 3) with both OTA and OTB concentrations of 5ng/mL, no analog OTB was detected. Whereas recovery was only 0.55% when the analog concentration was up to 250-fold. The prepared affinity monolithic column has strong affinity recognition capability and can effectively inhibit the nonspecific adsorption of coexisting interferents.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (7)
1. An affinity monolithic column of an ultra-high density modified aptamer of a hydrophobic matrix, which is characterized in that: the affinity monolithic column is an affinity monolithic column which is formed by polymerizing a quaternary ammonium salt strong cation functional monomer, a 2-oxazoline-5-ketone monomer and an acrylic ester cross-linking agent or a polysilsesquioxane cross-linking agent under the assistance of a pore-forming agent and an initiator through ultraviolet light to form a hydrophobic monolithic column matrix with the surface rich in positive charges of cations, and then self-assembling and adsorbing a nucleic acid aptamer sequence consisting of natural bases based on electrostatic action to form the hydrophobic matrix ultra-high density modified aptamer.
2. The affinity monolithic column of hydrophobic matrix ultra-high density modified aptamer of claim 1, wherein: the quaternary ammonium salt strong cation functional monomer is methacryloxyethyl trimethyl ammonium chloride DMC, the 2-oxazoline-5-ketone monomer is 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone VDMA, the acrylic cross-linking agent is ethylene glycol dimethacrylate EDMA, and the polysilsesquioxane cross-linking agent is methacrylic acid ester grafted cage-type oligomeric silsesquioxane POSS-MA.
3. The affinity monolithic column of hydrophobic matrix ultra-high density modified aptamer of claim 1, wherein: the pore-forming agent is a binary pore-forming agent consisting of N, N-dimethylformamide DMF and polyethylene glycol PEG-10000; the initiator is benzoin dimethyl ether DMPA.
4. An affinity monolithic column of a hydrophobic matrix ultra-high density modified aptamer according to any one of claims 1-3, wherein: the total mass percentage of the components is 100 percent, the content of the quaternary ammonium salt strong cation functional monomer DMC is 9.36 to 10.25 percent, the acrylic ester cross-linking agent EDMA is 7.28 to 7.97 percent, the content of the 2-oxazoline-5-ketone monomer VDMA is 4.16 to 4.55 percent, the content of N, N-dimethylformamide DMF is 67.96 to 71.94 percent, the content of polyethylene glycol PEG-10000 is 6.10 to 9.26 percent, and the content of the initiator benzoin dimethyl ether DMPA is 1.0 percent; or the quaternary ammonium salt strong cation functional monomer DMC is 7.52-8.32%, polysilsesquioxane cross-linking agent POSS-MA is 6.93-8.91%, 2-oxazoline-5-ketone monomer VDMA is 4.70-5.20%, N-dimethylformamide DMF is 71.95-73.77%, polyethylene glycol PEG-10000 is 6.26-6.42%, and initiator benzoin dimethyl ether DMPA is 1.0%.
5. The affinity monolithic column of hydrophobic matrix ultra-high density modified aptamer of claim 1, wherein: the nucleic acid aptamer is a natural base composition nucleic acid sequence GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA of the ochratoxin OTA.
6. A method of preparing an affinity monolith of the superhigh-density modified aptamer of a hydrophobic matrix according to any one of claims 1-3, wherein: the method comprises the following steps:
(1) Activation of anti-ochratoxin OTA nucleic acid aptamer:
centrifuging a natural base composition nucleic acid aptamer of the anti-ochratoxin OTA for 15 min at a rotation speed of 6000 r/min, adding 128 mu L of Tris-HCl buffer solution to dilute to 100 mu mol/L, carrying out vortex oscillation for 3 min, heating at 90 ℃ for 3 min, and annealing and cooling to room temperature for later use;
(2) Preparation of a polymerized monolithic column based on EDMA crosslinking:
weighing methacryloxyethyl trimethyl ammonium chloride DMC monomer, 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone VDMA monomer and ethylene glycol dimethacrylate EDMA cross-linking agent according to a proportion, adding pore-forming agent DMF and PEG-10000 and initiator DMPA, vortex oscillating for 3 min at room temperature, and performing ultrasonic degassing for 10 min to form a uniform solution; injecting the mixture into an alkenyl pretreated quartz capillary, sealing two ends, and placing the quartz capillary in 365 nm ultraviolet irradiation for 7 min for polymerization; after the reaction is completed, the prepared monolithic column is taken out and connected to a high-pressure solvent pump for a liquid chromatograph, and residues are removed by using methanol to obtain an EDMA cross-linked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges, which is hereinafter referred to as an EDV monolithic column;
(3) Preparation of a polymerized monolithic column based on POSS-MA crosslinking:
weighing methacryloxyethyl trimethyl ammonium chloride DMC monomer, 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone VDMA monomer and methacrylic acid ester grafted cage-type oligomeric silsesquioxane POSS-MA cross-linking agent according to a proportion, adding pore-forming agent DMF, PEG-10000 and initiator DMPA, carrying out vortex oscillation for 3 min at room temperature, and carrying out ultrasonic degassing for 10 min to form uniform solution; injecting the mixture into an alkenyl pretreated quartz capillary, sealing two ends, and placing the quartz capillary in 365 nm ultraviolet irradiation for 5 min for polymerization; after the reaction is completed, the prepared monolithic column is taken out and connected to a high-pressure solvent pump for a liquid chromatograph, and residues are removed by using methanol to obtain a POSS-MA cross-linked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges, which is hereinafter called PDV monolithic column;
(4) Preparation of an affinity monolithic column of a hydrophobic matrix ultra-high density modified aptamer:
and (3) filling the EDV monolithic column prepared in the step (2) or the PDV monolithic column prepared in the step (3) with Tris-HCl buffer solution, then injecting 20 mu L of the nucleic acid aptamer stock solution obtained in the step (1) through a liquid phase pump, and obtaining the hydrophilic monolithic column of the hydrophobic matrix ultrahigh-density modified aptamer, and storing at 4 ℃.
7. The method of manufacturing according to claim 6, wherein: the pH of the Tris-HCl buffer solution is 8.00, and the Tris-HCl buffer solution consists of 10mmol/L Tris-HCl,120 mmol/L NaCl and 5 mmol/L KCl.
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| CA2411827A1 (en) * | 2002-11-14 | 2004-05-14 | Mcmaster University | Method of immobilizing membrane-associated molecules |
| CN102940980A (en) * | 2012-10-22 | 2013-02-27 | 暨南大学 | Preparation method and application for hydrophilic organic polymer liquid phase monolithic chromatographic column |
| CN110102270A (en) * | 2019-06-03 | 2019-08-09 | 福州大学 | A kind of affine integral post of aptamer of specific recognition F2 toxin and preparation method thereof |
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