CN106399317B - Method for screening and characterizing single-stranded DNA aptamer of phthalate plasticizer - Google Patents
Method for screening and characterizing single-stranded DNA aptamer of phthalate plasticizer Download PDFInfo
- Publication number
- CN106399317B CN106399317B CN201610949635.5A CN201610949635A CN106399317B CN 106399317 B CN106399317 B CN 106399317B CN 201610949635 A CN201610949635 A CN 201610949635A CN 106399317 B CN106399317 B CN 106399317B
- Authority
- CN
- China
- Prior art keywords
- dbp
- screening
- stranded dna
- phthalate
- washing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000012216 screening Methods 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 30
- 108091023037 Aptamer Proteins 0.000 title claims description 57
- 108020004414 DNA Proteins 0.000 title claims description 35
- 102000053602 DNA Human genes 0.000 title claims description 34
- 108020004682 Single-Stranded DNA Proteins 0.000 title claims description 33
- 239000008029 phthalate plasticizer Substances 0.000 title claims description 13
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical class CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 claims abstract description 74
- 229920000936 Agarose Polymers 0.000 claims abstract description 42
- 239000004005 microsphere Substances 0.000 claims abstract description 41
- 238000012165 high-throughput sequencing Methods 0.000 claims abstract description 19
- 125000003277 amino group Chemical group 0.000 claims abstract description 15
- 238000005516 engineering process Methods 0.000 claims abstract description 14
- 238000012512 characterization method Methods 0.000 claims abstract description 10
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 9
- 108091008104 nucleic acid aptamers Proteins 0.000 claims abstract description 8
- 239000007790 solid phase Substances 0.000 claims abstract description 7
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000006482 condensation reaction Methods 0.000 claims abstract description 6
- 238000005191 phase separation Methods 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 26
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- YZBOVSFWWNVKRJ-UHFFFAOYSA-N Monobutylphthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(O)=O YZBOVSFWWNVKRJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000011324 bead Substances 0.000 claims description 17
- 239000012148 binding buffer Substances 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 238000000967 suction filtration Methods 0.000 claims description 11
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 11
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000005457 ice water Substances 0.000 claims description 8
- 239000004593 Epoxy Substances 0.000 claims description 7
- 239000007859 condensation product Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000002390 rotary evaporation Methods 0.000 claims description 7
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- AXPLQTKUQOWQLV-UHFFFAOYSA-N 5-[(2-methylpropan-2-yl)oxyamino]pentan-1-ol Chemical compound CC(C)(C)ONCCCCCO AXPLQTKUQOWQLV-UHFFFAOYSA-N 0.000 claims description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- 125000001931 aliphatic group Chemical group 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000012869 ethanol precipitation Methods 0.000 claims description 4
- 238000003760 magnetic stirring Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 108060002716 Exonuclease Proteins 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 102000013165 exonuclease Human genes 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 239000001257 hydrogen Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000001228 spectrum Methods 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 2
- LGRFSURHDFAFJT-UHFFFAOYSA-N Phthalic anhydride Natural products C1=CC=C2C(=O)OC(=O)C2=C1 LGRFSURHDFAFJT-UHFFFAOYSA-N 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 239000013504 Triton X-100 Substances 0.000 claims description 2
- 229920004890 Triton X-100 Polymers 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- JHIWVOJDXOSYLW-UHFFFAOYSA-N butyl 2,2-difluorocyclopropane-1-carboxylate Chemical compound CCCCOC(=O)C1CC1(F)F JHIWVOJDXOSYLW-UHFFFAOYSA-N 0.000 claims description 2
- 230000029087 digestion Effects 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 230000026731 phosphorylation Effects 0.000 claims description 2
- 238000006366 phosphorylation reaction Methods 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000011541 reaction mixture Substances 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims 1
- HEDRZPFGACZZDS-MICDWDOJSA-N deuterated chloroform Substances [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 claims 1
- 238000007865 diluting Methods 0.000 claims 1
- 230000010355 oscillation Effects 0.000 claims 1
- 238000005086 pumping Methods 0.000 claims 1
- 238000010791 quenching Methods 0.000 claims 1
- 230000000171 quenching effect Effects 0.000 claims 1
- 239000002002 slurry Substances 0.000 claims 1
- 239000007974 sodium acetate buffer Substances 0.000 claims 1
- 238000010025 steaming Methods 0.000 claims 1
- 239000004014 plasticizer Substances 0.000 abstract description 26
- 238000001514 detection method Methods 0.000 abstract description 18
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 abstract description 14
- 238000012360 testing method Methods 0.000 abstract description 13
- 235000013305 food Nutrition 0.000 abstract description 6
- -1 phthalic acid ester Chemical class 0.000 abstract description 5
- XNGIFLGASWRNHJ-UHFFFAOYSA-N o-dicarboxybenzene Natural products OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 abstract description 4
- 108091008102 DNA aptamers Proteins 0.000 abstract description 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 238000003757 reverse transcription PCR Methods 0.000 description 40
- IRIAEXORFWYRCZ-UHFFFAOYSA-N Butylbenzyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCC1=CC=CC=C1 IRIAEXORFWYRCZ-UHFFFAOYSA-N 0.000 description 28
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 description 27
- 239000012085 test solution Substances 0.000 description 10
- 239000000523 sample Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 238000005498 polishing Methods 0.000 description 6
- 238000011529 RT qPCR Methods 0.000 description 5
- 229940125782 compound 2 Drugs 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 239000010931 gold Substances 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- MQIUGAXCHLFZKX-UHFFFAOYSA-N Di-n-octyl phthalate Chemical compound CCCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCCC MQIUGAXCHLFZKX-UHFFFAOYSA-N 0.000 description 4
- 229940126214 compound 3 Drugs 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229910001385 heavy metal Inorganic materials 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 230000009897 systematic effect Effects 0.000 description 3
- ZVFDTKUVRCTHQE-UHFFFAOYSA-N Diisodecyl phthalate Chemical compound CC(C)CCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCCC(C)C ZVFDTKUVRCTHQE-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000002484 cyclic voltammetry Methods 0.000 description 2
- HBGGXOJOCNVPFY-UHFFFAOYSA-N diisononyl phthalate Chemical compound CC(C)CCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCC(C)C HBGGXOJOCNVPFY-UHFFFAOYSA-N 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 description 2
- 229960001031 glucose Drugs 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000007885 magnetic separation Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002957 persistent organic pollutant Substances 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- JQCXWCOOWVGKMT-UHFFFAOYSA-N phthalic acid diheptyl ester Natural products CCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCC JQCXWCOOWVGKMT-UHFFFAOYSA-N 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920000915 polyvinyl chloride Polymers 0.000 description 2
- 239000004800 polyvinyl chloride Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 101100317222 Borrelia hermsii vsp3 gene Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000004803 Di-2ethylhexylphthalate Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 231100000507 endocrine disrupting Toxicity 0.000 description 1
- 239000000598 endocrine disruptor Substances 0.000 description 1
- 231100000049 endocrine disruptor Toxicity 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000005003 food packaging material Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008266 hair spray Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- MINVSWONZWKMDC-UHFFFAOYSA-L mercuriooxysulfonyloxymercury Chemical class [Hg+].[Hg+].[O-]S([O-])(=O)=O MINVSWONZWKMDC-UHFFFAOYSA-L 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- APVPOHHVBBYQAV-UHFFFAOYSA-N n-(4-aminophenyl)sulfonyloctadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 APVPOHHVBBYQAV-UHFFFAOYSA-N 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000003021 phthalic acid derivatives Chemical class 0.000 description 1
- 150000003022 phthalic acids Chemical class 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 231100000527 sperm abnormality Toxicity 0.000 description 1
- 230000019100 sperm motility Effects 0.000 description 1
- 238000000141 square-wave voltammogram Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- ZZORFUFYDOWNEF-UHFFFAOYSA-N sulfadimethoxine Chemical compound COC1=NC(OC)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ZZORFUFYDOWNEF-UHFFFAOYSA-N 0.000 description 1
- 229960000973 sulfadimethoxine Drugs 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1048—SELEX
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a phthalic acid ester plasticizer DNA aptamer, a screening and characterization method thereof and an electrochemical sensor. The method of the invention comprises the following steps: the method comprises the following steps: preparing a dibutyl phthalate derivative having an amino group at the terminal of one of linear fatty side chains by chemical synthesis; step two: covalently attaching DBP-NH2 to the agarose microspheres by condensation reaction of amino groups with oxirane activating groups on the agarose microspheres; step three: performing solid phase separation-based screening of nucleic acid aptamers; step four: after screening, carrying out high-throughput sequencing on the obtained enrichment library; step five: performing affinity and selectivity tests on two sequences DBP-1 and DBP-2 with the highest occurrence frequency in high-throughput sequencing; step six: and constructing the electrochemical sensor based on the DBP-1. The invention can be used for developing various detection technologies and biosensors, and has important application value for convenient detection of phthalate plasticizers in foods or environments.
Description
Technical Field
The invention relates to a single-stranded DNA aptamer of a phthalate plasticizer, a screening and characterization method thereof and an electrochemical sensor using the single-stranded DNA aptamer, belonging to the technical field of biology.
Background
Phthalate Esters (Phthalic acids or Phthalic Acid Esters, PAEs) are a generic name for Esters of Phthalic Acid, whose chemical structure is composed of a planar aromatic hydrocarbon and two aliphatic side chains. PAEs are mainly used for polyvinyl chloride materials, so that polyvinyl chloride is changed from hard plastic into elastic plastic, and the plasticizing effect is achieved. PAEs are widely used in hundreds of products such as toys, food packaging materials, medical blood bags and hoses, vinyl floors and wallpaper, cleaners, lubricants, personal care products (e.g., nail polish, hair sprays, soaps, and shampoos).
PAEs are Persistent Organic Pollutants (POPs) which are difficult to degrade in ester compounds, and are also recognized environmental endocrine disruptors. PAEs are a class of fat-soluble substances that can enter the human body through respiration, diet, and skin contact, causing harm to the human body. Researches show that the PAEs play a role similar to estrogen in human bodies and animal bodies, can interfere endocrine, reduces the sperm quantity and the number of sperms of men, has low sperm motility, abnormal sperm morphology and severe testicular cancer, and is the main culprit for causing the reproduction problem of the men. It also increases the chances of women suffering from breast cancer and can also harm their reproductive system of future-bearing babies in men. The PAEs currently considered as harmful substances are 15, of which 6, di (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), di-n-octyl phthalate (DNOP), Butyl Benzyl Phthalate (BBP), diisononyl phthalate (DINP), diisodecyl phthalate (DIDP), which have been classified by the european union as substances that are harmful to humans and the environment whose total amount must be less than 1%. China has clear limit values for various PAEs in the pollutant discharge standards of domestic drinking water, surface water and sewage treatment plants. DBP has the lowest limit due to its greatest potential endocrine disrupting toxicity. For example, the limit of the sanitary standard for domestic drinking water (GB5749-2006) in China to DBP is as low as 0.003mg/L (1nM, 3 ppb). At present, the detection of the plasticizer at home and abroad is only limited by large instruments such as high performance liquid chromatography and mass spectrometry, and the on-site high-efficiency and timely monitoring cannot be realized. How to rapidly, accurately, directly and effectively detect the plasticizer in food and water is a technical problem which is expected to be solved by relevant scientific researchers such as environment, food safety and the like.
Aptamers are single-stranded or double-stranded DNA or RNA obtained by SELEX technology (Systematic Evolution of Ligands by Expo-netional engineering, i.e.systematic exponential Enrichment of aptamer phylogenetic techniques) (Nature,1990,346, 818-. The aptamer can specifically recognize various target molecules including proteins, small molecules, cells and tissues, has high stability, easy synthesis and modification and low cost, and has wide application prospects in the fields of biosensing, imaging, drug research and development and the like. At present, many aptamers are obtained by an in vitro screening technology, and many aptamers can be detected quickly and sensitively on line, but reports for specifically recognizing the aptamers of the phthalate plasticizer are not seen.
Disclosure of Invention
The invention aims to provide a single-stranded DNA aptamer of a phthalate plasticizer, a screening and characterization method thereof and an electrochemical sensor using the single-stranded DNA aptamer. The invention screens out a single-stranded DNA aptamer sequence which can be combined with the phthalate plasticizers such as DBP and the like in specificity and high affinity by utilizing SELEX technology, and constructs an electrochemical sensor which can carry out high-sensitivity and high-specificity detection on the phthalate plasticizers such as DBP and the like by utilizing the aptamer.
The single-stranded DNA aptamer with high specificity and high affinity with the phthalate plasticizer provided by the invention. The aptamer has highly enriched single-stranded DNA sequence from the same C-rich base family 5' CTTTCTGTCCTTCCGTCACN1-4TCCCACGCATTCTCCACAT3' and/or a single or double base variant thereof. Wherein the two aptamers with the highest occurrence frequency in high-throughput sequencing are respectively: DBP-1: 5' CTTTCTGTCCTTCCGTCACATCCCACGCATTCTCCACAT3', and DBP-2:5' CTTTCTGTCCTTCCGTCACAGGTCCCACGCATTCTCCACAT3 '.
The invention provides a method for screening and characterizing a single-stranded DNA aptamer of a phthalate plasticizer and an electrochemical sensor using the single-stranded DNA aptamer, which comprises the following steps: the method comprises the following steps: dibutyl phthalate (DBP) derivative (DBP-NH) having an amino group at the end of one of the linear aliphatic side chains was prepared by chemical synthesis2) (ii) a Step two: DBP-NH is subjected to condensation reaction by amino and ethylene oxide activated group on agarose microspheres2Covalently attached to agarose microspheres; step three: performing a SELEX screen of DNA aptamers based on solid phase separation; step four: after screening, carrying out high-throughput sequencing on the obtained enrichment library; step five: performing affinity and selectivity tests on two sequences DBP-1 and DBP-2 with the highest occurrence frequency in high-throughput sequencing; step six: and constructing the electrochemical sensor based on the DBP-1.
The invention also provides an electrochemical biosensor based on the aptamer, wherein a signal probe of the electrochemical biosensor is DBP-1-10T-C-Fc or DBP-1-C-Fc.
The invention prepares a dibutyl phthalate (DBP) derivative (DBP-NH) with an amino group at the terminal of one linear fatty side chain by chemical synthesis2). Then carrying out condensation reaction on amino and ethylene oxide activated groups on the agarose microspheres to obtain DBP-NH2Covalently attached to agarose microspheres. Followed by solid phase separation based screening for nucleic acid aptamers. The enriched library obtained after 4 rounds of screening was subjected to high throughput sequencing, and the first 100 DNA sequences with the highest frequency of occurrence were all from the same family. The real-time quantitative PCR technology is utilized to carry out the reaction on 2 aptamers (DBP-1 and DBP-2) with the highest frequency of occurrence on DBP-NH2The affinity of (a) was tested at 70. + -.5 nM and 100. + -.5 nM, respectively. Affinity comparison of DBP-1 and DBP-2 to DBP, DEHP, BBP versus DBP-NH2The affinity of (A) is 1.5-4.0 times higher. DBP-1 and DBP-2 have good specificity to DBP, DEHP and BBP, and have good specificity to glucose, kanamycin, ethanol and other small molecules and heavy metal ions (Hg)2+,Pb2+,Ni2+,Cd2+) There is no significant affinity. The DBP electrochemical sensor is constructed based on the aptamer. The sensor has good selectivity and sensitivity to DBP. The detection limit reaches 10pM, and the kinetic interval is 10pM-1 mM. Due to the structural similarity of phthalate plasticizers, it is expected that the aptamers of the invention should also have high affinity and selectivity for other phthalate plasticizers.
The invention has the following characteristics and technical advantages:
(1) the single-stranded DNA aptamer sequence obtained by the invention has high affinity (dissociation constant K) with DBP, BBP and DEHPdRespectively in the nM range), and has good specificity. Due to the structural similarity of phthalate plasticizers, it is expected that the aptamers of the invention should also have high affinity and selectivity for other phthalate plasticizers.
(2) The solid phase immobilization method of the target molecule is to firstly carry out chemical modification on the hydrophobic side chain of DBP to obtain DBP-NH2And then DBP-NH is reacted by condensation reaction between amino group and epoxy group2Covalently linked to ethylene oxide activated agarose microspheres such that the phthalate diester groups are exposed on the surface of the microspheres, allows for the screening of phthalate plasticizers common functional groups, rather than just one specific phthalate plasticizer.
(3) The target molecule solid phase fixing mode mentioned in (2) can also greatly reduce the non-specific adsorption of the interface and reduce the number of screening rounds, and the invention combines with high-throughput sequencing technology to obtain the highly enriched aptamer through 4 rounds of screening.
(4) The nucleic acid aptamer primer region sequence of the phthalate plasticizer does not participate in the combination with target molecules, so that the complex work of engineering design can be saved, the cost of the sensor can be greatly reduced and the sensor design can be simplified due to the short length of the probe, and the application of the nucleic acid aptamer is very convenient.
(5) The nucleic acid aptamer sequence can be combined with various sensing platforms including electrochemical sensors to realize high-sensitivity and high-specificity detection of phthalate plasticizers; compared with a detection technology based on a large instrument, the preparation method of the sensor based on the aptamer is simple, the operation is more convenient, the cost is low, and the sensor is suitable for field detection.
The invention utilizes the in vitro screening technology (SELEX: ligand systematic evolution technology of exponential enrichment) of the aptamer to screen and obtain the single-stranded DNA aptamer with high specificity and high affinity with the phthalate plasticizer from a chemically synthesized random DNA library. Lays a foundation for the construction of a new method for detecting the phthalate plasticizer in foods and environments.
The invention prepares a dibutyl phthalate (DBP) derivative (DBP-NH) with an amino group at the terminal of one linear fatty side chain by chemical synthesis2). Then carrying out condensation reaction on amino and ethylene oxide activated groups on the agarose microspheres to obtain DBP-NH2Covalently attached to agarose microspheres. Followed by solid phase separation based screening for nucleic acid aptamers. The enriched library obtained after 4 rounds of screening was subjected to high throughput sequencing, and the first 100 DNA sequences with the highest frequency of occurrence were all from the same family. The real-time quantitative PCR technology is utilized to perform quantitative PCR on 2 aptamers with the highest occurrence frequency, namely DBP-1 and DBP-2 and DBP-NH2The affinity of (a) was tested at 70. + -.5 nM and 100. + -.5 nM, respectively. Affinity comparison of DBP-1 and DBP-2 to DBP, DEHP, BBP versus DBP-NH2The affinity of (A) is 1.4-4.0 times higher. Both DBP-1 and DBP-2 have good specificity, and the relative affinity is 1100 times higher than that of representative interferents such as glucose, kanamycin, ampicillin and ethanol. The DBP electrochemical sensor is constructed based on the aptamer. The sensor has good selectivity and sensitivity to DBP. The detection limit reaches 10pM, and the kinetic interval is 10pM-1 mM. Due to the structural similarity of phthalate plasticizers, it is expected that the aptamers of the invention should also have high affinity and selectivity for other phthalate plasticizers. The invention makes up the vacancy that no aptamer of the phthalate ester compound exists. As a compound having high affinity andspecific biological recognition, the nucleic acid aptamers of the invention can be used to develop a wide variety of detection techniques and biosensors. Has important application value for convenient detection of phthalate plasticizer in food or environment.
Drawings
FIG. 1 is a technical scheme of the present invention for screening single-stranded DNA aptamers using phthalate plasticizers.
FIG. 2 shows DBP-NH in the present invention2Synthetic route maps of (1).
FIG. 3 is a one-dimensional nuclear magnetic hydrogen spectrum of DBP-NH-Boc (Compound 2) in the present invention.
FIG. 4 shows DBP-NH in the present invention2Electrospray mass spectrometry characterization data for (compound 3).
FIG. 5 is a graph showing the respective characterization of two aptamers (DBP-1 and DBP-2) with the highest enrichment efficiency by fluorescence inverted microscope according to the present invention on DBP-NH2Fluorescence pictures of affinity and selectivity of modified agarose microspheres: (a) unmodified DBP-NH2The agarose microspheres of (4); (b) unmodified DBP-NH after incubation with DBP-1-FAM and DBP-2-FAM2The agarose microspheres of (4); (c) DBP-NH incubated with DBP-1-FAM and DBP-2-FAM2Modified agarose microspheres.
FIG. 6 shows the measurement of DBP-NH by real-time quantitative PCR in the present invention for DBP-1(a) and DBP-2(b)2Data of affinity of (a).
FIG. 7 shows the present invention using real-time quantitative PCR for DBP-1(a) and DBP-2(b) versus DBP-NH2BBP, DBP, DEHP and relative affinity test data for the selective test solution, the relative affinity value being the number of DBP-1-RT-PCRs or DBP-2-RT-PCRs that compete with each test sample divided by the number of DBP-1-RT-PCRs or DBP-2-RT-PCRs that compete in the presence of the selective test solution.
FIG. 8 is a schematic diagram of an electrochemical sensor constructed based on DBP-1 according to the present invention (a), square wave voltammograms (b, c) for detecting 16 PAE mixed standards at different concentrations, and heavy metal ions (Hg)2+,Pb2+,Ni2+) And antibiotics (10. mu.M kanamycin and 10. mu.M sulfadimethoxine mix samples)And (d) testing the selectivity. Wherein (b) and (C) correspond to sensors using DBP-1-10T-C-Fc and DBP-1-C-Fc as signaling probes, respectively.
Detailed Description
Table 1: nucleic acid probe sequences used in the present invention (underlined sequences in the table are regions to which primers bind in PCR)
TABLE 2 first 100 nucleic acid sequences of the present invention (5'-3') which were most frequently found by high-throughput sequencing
Sequences are ordered from high to low frequency of occurrence in high throughput sequencing. The underlined portion of the first sequence is a highly enriched conserved sequence. The other sequences are underlined in bold and the variant compared to sequence 1.
Example 1 technical route for screening Single-stranded DNA aptamers to phthalate plasticizers Using SELEX and high throughput sequencing
As shown in FIG. 1, the technical route of screening single-stranded DNA aptamers of phthalate plasticizers by SELEX and high-throughput sequencing technology of the invention comprises the following steps: (1) DBP-NH2Chemical synthesis and characterization of (1); (2) DBP-NH2Agarose microspheres activated with Epoxy (Epoxy-activated Sepharose)TM6B) Coupling and characterization of (1); (3) will be 6X 1014(1nmol) of a commercially synthesized starting single-stranded DNA library (pool)0TABLE 1) and DBP-NH2Mixing and incubating the coupled agarose microspheres; (4) will be combined with a coreSeparating and washing the agarose microspheres of the acid aptamer and DNA sequences which are not combined with the agarose microspheres; (5) carrying out hot elution on the aptamer on the washed agarose microspheres; (6) reverse Primer (PO) modified by 5-terminal phosphate radical4-RP-SELEX, table 1) and forward primer (FP-SELEX, table 1), subjecting the eluted aptamer to PCR amplification; (7) carrying out lambda exonuclease reaction to degrade the phosphate radical modified complementary strand, preparing a single-stranded DNA library at low cost, and enabling the library to enter the next screening cycle; (8)4 rounds of SELEX-following library (pool)4) Performing high-throughput sequencing; (9) the two aptamers DBP-1 and DBP-2 with the highest frequency of occurrence were characterized for affinity and selectivity.
Example 2 amino derivative of DBP (DBP-NH)2) Synthesis and characterization of
DBP-NH2The synthetic route of (2) is shown in FIG. 2. In the embodiment, all the reagents are analytically pure, the reaction processes in each step are monitored in real time by using thin-layer chromatography, and the product is separated and purified by using a silica gel column. The specific synthesis method, conditions and characteristics are as follows (1-3):
(1) preparation of monobutyl phthalate (Compound 1)
To a 50 ml dry round bottom flask containing phthalic anhydride (8.5g, 57mmol) and anhydrous treated tetrahydrofuran (7.5 ml) and clean dry magnetite was added n-butanol (5 ml) and heated to reflux for 9 hours at 60 ℃ with magnetic stirring. An insoluble white solid formed. After standing at room temperature, the mixture was filtered, and the bottle was washed with tetrahydrofuran and filtered again. And (5) carrying out rotary evaporation to obtain a white pasty substance. Adding CH2Cl2After that, there was a white precipitate. Filtering, rotary evaporating to obtain oily liquid. Adding 100 ml deionized water, extracting with equal volume of ethyl acetate for 3 times, extracting organic phase with saturated NaCl for 1 time, and adding anhydrous Na2SO4The organic phase was dried and left overnight. The mixture was filtered, spun-dried to obtain 10.986g, and the residue was purified by silica gel column separation (dichloromethane/methanol (95:5) as a mobile phase). After spin-drying, vacuum was applied to obtain 2.1g of monobutyl phthalate (Compound 1). High performance liquid chromatography (Agilent 1200) separated as a single peak and the product was pure.
(2) Preparation of DCC condensation product (Compound 2)
In this experiment, monobutyl phthalate (compound 1) and 5- (N-tert-butoxyamino) -1-pentanol are fed in a ratio of 1: 1.2. To 20mL of anhydrous tetrahydrofuran was added monobutyl phthalate (1.158g), and dicyclohexylcarbodiimide (DCC, 5.495g) was added under magnetic stirring and ice-water bath. 4-dimethylaminopyridine (DMAP, 0.217g) was added after 10 minutes and 5- (N-tert-butoxyamino) -1-pentanol (1.723g, dissolved in 5ml of anhydrous tetrahydrofuran, added dropwise) was added after 20 minutes. After stirring in the ice-water bath for 1 hour, the ice-water bath was removed and the reaction was carried out at room temperature for 24 hours. And (3) carrying out suction filtration on the reaction solution, washing the flask with anhydrous tetrahydrofuran, carrying out suction filtration, and carrying out rotary evaporation on the filtrate. 20ml of petroleum ether is added for washing and suction filtration is carried out. Placing in a refrigerator for suction filtration overnight, washing a flask with petroleum ether for suction filtration, and performing rotary evaporation. The product was then purified by column on silica gel (eluent petroleum ether: ethyl acetate 4:1(v: v)), and rotary evaporated. After purification, approximately 0.5g of DCC condensation product (Compound 2) was obtained. One-dimensional nuclear magnetic hydrogen spectrum characterization (VNMRS-600 MHz, TMS as internal standard, CDCl)3As solvent) confirmed to give the expected compound 2 (fig. 3).
(3) DBP derivative (DBP-NH) modified by alkyl chain terminal amino2) Preparation of (Compound 3)
The DCC condensation product (compound 2) (0.5g), dichloromethane (DCM, 2 ml) and trifluoroacetic acid (TFA, 2 ml) were added to a three-necked flask and magnetically stirred at room temperature for 40 minutes. The mixture was then filtered and rotary evaporated to give an oily liquid. The oily liquid was extracted three times with ethyl acetate and saturated NaHCO3Washed once and dried over anhydrous sodium sulfate. Finally, filtration and rotary evaporation gave 0.1g of DBP-NH2. Electrospray mass spectrometry (Agilent LC/SMD TOF) showed molecular ion peak 308.1856 with Compound 3 (C)17H25O4N) (fig. 4), indicating the successful preparation of compound 3.
Example 3 DBP-NH2Coupling on epoxy-activated agarose microspheres
0.1g of epoxy-activated agarose microspheres was swollen with deionized water and washed repeatedly with 20mL of deionized water to obtain 350. mu.L of wet spheres. Then 0.2M Na was added2CO3(pH. apprxeq.12) agarose beads were washed. To 500. mu.L of the reaction system, 46.7mg (0.15mmol) of DBP-NH was added2The reaction mixture was left at room temperature for 48 hours with shaking. After the reaction was completed, a buffer solution of sodium acetate (0.1M sodium acetate, 0.5M NaCl) having a pH of 4.5 and a buffer solution of sodium carbonate (0.2M NaHCO) having a pH of 12 were added3/Na2CO30.5M NaCl) was repeatedly and alternately washed three times, finally washed with water, and the volume was constant to 500 μ L, and stored in a refrigerator at 4 ℃ for later use. Elemental analysis (Elementar Vario MicroUBE, Germany) showed that the agarose beads contained 33.61%, 9.26%, 0.92% and 1.09% of each of the constituent elements C, H, N and S, respectively. Coupling of DBP-NH2Then, the proportions of the constituent elements C, H, N, S were 50.21%, 7.88%, 2.3% and 0.9%, respectively, wherein the large increase in the proportions of C and N after coupling confirmed DBP-NH2Successfully coupled to epoxy activated agarose microspheres.
Example 4 SELEX screening
Random single-stranded DNA library (Pool) used for screening in the invention0Table 1) is synthesized by Biotechnology engineering (Shanghai) Ltd and purified by polyacrylamide gel electrophoresis (PAGE). Pool0The total length is 80 bases, and the sequence comprises a fixed sequence with the length of 20 bases at two ends and a random sequence with the length of 40 bases in the middle. The immobilized sequences were related to the upstream primer (FP-SELEX, Table 1) and the downstream Primer (PO) in the PCR step of SELEX, respectively4-binding region of RP-SELEX, table 1). The upstream primer and the downstream primer are both synthesized by TaKaRa, Takara, Inc. Wherein the 5' end of the downstream primer is subjected to phosphorylation modification. Pool0FP-SELEX and PO4Each of-RP-SELEX was prepared into 100. mu.M stock solutions with 1 XTTris-EDTA buffer (10mM Tris,1mM EDTA, pH8.0) and stored at-80 ℃ until use.
First screening: 1nmole of Pool0(100. mu.M stock, 10. mu.L) was diluted in 490. mu.L of binding buffer BB (20mM Tris, 100mM sodium chloride, 2mM magnesium chloride, 5mM potassium chloride, 1mM calcium chloride, 1% Tween20, 0.03% triton X-100, 2% DMSO, pH 7.9). The solution was heated at 95 ℃ for 10 minutes, quenched in an ice-water bath for 5 minutes, and allowed to stand at room temperature. The ligation prepared in example 3 was performedDBP-NH2The agarose microspheres (100. mu.L) were washed three times with binding buffer BB. Then adding the above-mentioned heat-treated pool into the above-mentioned washed agarose microsphere0The solution was incubated at room temperature for 1 hour with rotation. Separating with 10K ultrafiltration tube, washing agarose ball with binding buffer BB for three times, adding 100 μ L binding buffer BB, heating at 90 deg.C under shaking for ten minutes, separating with ultrafiltration tube, and collecting supernatant. The eluate was subjected to PCR amplification, and the total volume of the reaction was 2 mL. The obtained PCR product was qualitatively characterized by PAGE and purified by ethanol precipitation. Then preparing single-stranded DNA by using Lamda exonuclease digestion method, and purifying by using ethanol precipitation method to obtain enriched single-stranded DNA library Pool1。
Rounds 2-4 SELEX: and (3) redissolving the Pool obtained by the SELEX in the first round, quantifying, and putting about 300pmol for the next round of screening, wherein the specific screening process and conditions are the same as those of the first round of screening. Finally obtaining the enriched single-stranded DNA library Pool4。
Example 5 Pool4High throughput sequencing analysis of
Adding Pool4Sent to Beijing Nuo-He biogenic bioinformatics science and technology company for high-throughput sequencing. High throughput sequencing results (Table 2) showed that the top 100 most frequently occurring single stranded DNA sequences were highly enriched and were from the same family (5' CTTTCTGTCCTTCCGTCACN)1-4TCCCACGCATTCTCCACAT3') and is rich in C bases. Wherein the first 2 single-stranded DNA sequences with the highest frequency of occurrence are respectively:
DBP-1: 5'CTTTCTGTCCTTCCGTCACATCCCACGCATTCTCCACAT3' (39 bases)
DBP-2:5'CTTTCTGTCCTTCCGTCACAGGTCCCACGCATTCTCCACAT3' (41 bases)
The two aptamers differ by only 2 bases (underlined).
Example 6 measurement of DBP-1 and DBP-2 vs DBP-NH Using fluorescence inverted microscope2Affinity and selectivity of modified agarose microspheres
DBP-1 and DBP-2 (1. mu.M, DBP-1-FAM and DBP-2-FAM, Table 1, Shanghai Biotech Co., Ltd.) modified with 5' -terminal fluorophore FAM were mixed with blank and 10. mu.M, respectivelyLDBP-NH2The modified agarose microspheres were incubated in 200. mu.L of binding buffer BB for 1 hour, washed, placed on a clean glass slide, observed using a fluorescence inverted microscope and photographed.
As shown in FIG. 5, the agarose microspheres themselves are very weak in fluorescence (a); the blank agarose microspheres incubated with DBP-1-FAM and DBP-2-FAM also showed weak fluorescence (b); DBP-NH incubated with DBP-1-FAM and DBP-2-FAM2The modified agarose microspheres all showed strong fluorescence brightness (c). The experimental results show that DBP-1 and DBP-2 are both on DBP-NH2The modified agarose microspheres have stronger affinity and are selectively combined with DBP-NH on the surfaces of the microspheres2Bound, not bound to the agarose microsphere substrate. Wherein DBP-NH is incubated with DBP-1-FAM2The fluorescence brightness rate of the modified agarose microspheres is higher than DBP-NH incubated with DBP-2-FAM2Modified agarose microspheres, indicating DBP-1 vs DBP-NH2Has slightly higher affinity than DBP-2. The experimental results also show that DBP-1 and DBP-2 can react with DBP-NH without the participation of primer region sequences of Pool2Has good affinity and selectivity, thus not only saving the complex work of engineering design, but also greatly reducing the cost of the sensor and simplifying the sensor design due to the short length of the probe, and being very convenient for the application of the aptamer.
Example 7 measurement of DBP-1 and DBP-2 vs DBP-NH by real-time quantitative PCR technique (RT-PCR)2Affinity of (2)
(1) Design and synthesis of aptamer probes for affinity testing
In this example, RT-PCR technique was used to quantify DMP-NH2The number of aptamers bound to the modified magnetic beads. For this, primer regions for PCR amplification were added to both ends of DBP-1 and DBP-2, respectively, to obtain two sequences, DBP-1-RT-PCR and DBP-2-RT-PCR (Table 1, Shanghai Biotechnology Co., Ltd.). It is worth noting that the primer region sequences used in this assay are different from those used in the SELEX screening process, and if the results of the test indicate that the aptamers still have affinity, it is sufficient to demonstrate that DBP-1 and DBP-2 are able to target DBP-NH without the help of Pool primer region sequences2Has good affinityForce and selectivity.
(2)DBP-NH2Preparation of modified magnetic beads
(3) Affinity (dissociation constant K)d) Measurement of (2)
DBP-1-RT-PCR or DBP-2-RT-PCR (1pM, 10pM, 100pM, 1nM, 10nM, 50nM, 75nM, 100nM, 200nM, 300nM) was configured at different concentrations in binding buffer BB. Adding equal amount of DBP-NH prepared in the step (2) into the aptamer solutions respectively2Modified M-270 magnetic beads (10. mu.L) were incubated at room temperature for 30 minutes. After 4 minutes on a strong magnet, the supernatant was removed. Wash 4 times with 200. mu.L of binding buffer. Add 100 u L binding buffer BB, in 90 degrees C shaking and heating ten minutes after magnetic separation, collect the supernatant. The supernatant was subjected to RT-PCR. The amount of aptamer bound to the magnetic beads was determined using different concentrations of aptamer according to a standard curve. The amount of DBP-1-RT-PCR or DBP-2-RT-PCR bound to the magnetic beads was plotted against the concentration of DBP-1-RT-PCR or DBP-2-RT-PCR dosed (FIG. 6). According to the aptamer with DBP-NH2Is a 1:1 combinationProportional, calculating K by non-linear fittingdThe values are respectively: 70. + -.5 nM (DBP-1-RT-PCR, FIG. 6a) and 100. + -.5 nM (DBP-2-RT-PCR, FIG. 6b), errors were from three replicates. And starting the library pool0To DBP-NH2The modified magnetic beads have no affinity.
Example 8 testing of DBP-1 and DBP-2 on DBP-NH by RT-PCR2Relative affinity testing for BBP, DBP, DEHP and selectivity for possible interferents
Since plasticizers present in the environment are not modified with amino groups and are of a large variety, it is necessary to test whether the aptamers selected have a good affinity and selectivity for these plasticizers. To this end we performed the following competition assay to determine DBP-NH2Relative affinity tests for BBP, DBP, DEHP and selectivity for possible interferents.
The same volume of DBP-1RT-PCR or DBP-2 RT-PCR (5. mu.L, 10. mu.M) at the same concentration was mixed with the same amount (10. mu.L) of DBP-NH, respectively2The modified magnetic beads were incubated in 500. mu.L of the binding buffer for 1 hour, and then washed 3 times with 100. mu.L of the binding buffer. The washed magnetic beads were mixed with 120. mu.L of 10. mu.M DBP-NH, respectively2DBP, DEHP, BBP or selective test solutions containing various types of small molecules were incubated in a mixture for 1 hour. The selective test solution contains representative interferents such as glucose, kanamycin, ampicillin, ethanol, etc., and the concentration of each interferent is 10 μ M. After 4 minutes on a strong magnet, the supernatant was removed. And performing RT-PCR on the amount of DBP-1-RT-PCR or DBP-2-RT-PCR in the supernatant, and determining the amount of DBP-1-RT-PCR or DBP-2-RT-PCR in the supernatant according to a standard curve.
In the above experiment, DBP-1-RT-PCR or DBP-2-RT-PCR was first performed on DBP-NH2Modified magnetic beads with bound DBP-NH subsequently added2Or DBP or DEHP or BBP or a selective test solution with DBP-NH2The modified magnetic beads compete for part of the DBP-1-RT-PCR or DBP-2-RT-PCR with DBP-NH in solution2Or DBP or DEHP or BBP or a component of the selective test solution, into the solution. The amount of DBP-1-RT-PCR or DBP-2-RT-PCR in the supernatant thus reflects the amount of DBP-NH2Or DBP or DEHP or BBP or selective test solutions for relative affinity.
As shown in FIG. 7, for the purpose of simple and intuitive expression, the relative number of DBP-1-RT-PCR or DBP-2-RT-PCR competed by the selective test solution is 1, i.e., the value of the relative affinity is the number of DBP-1-RT-PCR or DBP-2-RT-PCR competed by each test sample divided by the number of DBP-1-RT-PCR or DBP-2-RT-PCR competed in the presence of the selective test solution. Plotting DBP-1(a) and DBP-2(b) against DBP-NH2BBP, DBP, DEHP relative affinities. DBP-1-RT-PCR or DBP-2-RT-PCR on DBP-NH2Or the relative affinity of DBP, DEHP or BBP is about 240-1100 times higher than that of the selective test solution, which indicates that the aptamer screened by the invention has good selectivity. In addition, the relative affinities of DBP-1-RT-PCR and DBP-2-RT-PCR to DBP, DEHP and BBP were compared to DBP-NH2The affinity of (A) is about 1.4 to 4.0 times higher. It is demonstrated that the aptamers screened according to the invention have a very good affinity for plasticizers, even higher than the affinity of the amino-modified DBP used in the screening. The results show that the aptamer screened by the invention has high affinity and specificity to common plasticizers, which lays the foundation of molecular recognition for the construction of a novel method for detecting the plasticizer based on the aptamer and has important significance for the development of detection technologies of the plasticizer in the environment and food.
Example 9 construction of aptamer DBP-1 based plasticizer electrochemical biosensor and detection of plasticizer
(1) Cleaning of disk gold electrode surface
The gold disk electrode (diameter 2mm) was rinsed with ultrapure water, and 1 μm, 0.3 μm, and 0.05 μm of Al were used in this order2O3Polishing the surface of the electrode by using polishing powder (adding a small amount of ultrapure water and solid powder on polishing cloth for polishing for 5-10 minutes), washing the surface by using ultrapure water after each polishing, performing ultrasonic treatment in the ultrapure water for 5 minutes, and then performing the next polishing step. The smooth polished electrode is connected with a VMP3 multi-channel electrochemical workstation through a three-electrode system at 0.5M H2SO4In the range of-0.4-1.2V and taking 100mV/s as cyclic voltammetry scanning for 36 circles (taking a gold electrode as a working electrode and a saturated mercurous sulfate electrode as a reference electrode)Electrode, platinum electrode as counter electrode) until the cyclic voltammogram is substantially stable. If no obvious corresponding redox peak is observed, the gold electrode is polished again by the above steps.
(2) Construction of plasticizer electrochemical sensor (SD-EAB) based on signal probe chain substitution
mu.L of a PBS/1M NaCl solution (1 XPB, 1M NaCl, pH 7.4) containing 0.5. mu.M HS-DBP-1 (Table 1) and 0.5. mu.M of a 3' -end modified redox group ferrocene (Fc) or DBP-1-C-Fc (Table 1) was heated in a water bath at 95 ℃ for 10min and slowly cooled to room temperature. Then, 1. mu.L of TCEP (10mM) was added and the mixture was left at room temperature for 1 hour. And (3) putting the disc gold electrode cleaned in the step (1) into the solution, and assembling at room temperature overnight. Washed three times with PBS/1M NaCl solution, and the electrode was placed in 100. mu.L of 1mM [ S (CH)2)2(OCH2CH2)6OCH3]2(OEG6OMe) in PBS/1M NaCl for 1 hour at room temperature. Three washes with PBS/1M NaCl solution were performed, and finally left to equilibrate in binding buffer BB for 1 hour, and SWV was swept to obtain a stable baseline for use.
(3) Detection and selectivity testing of PAE
Mixing 16 PAE standard solutions (Chem Service, each containing 10ppm) or 10 μ M standard solution containing different heavy metal ions (Hg)2+,Pb2+,Ni2+,Cd2+) After 1 hour incubation with the electrode, the binding buffer solution BB of (3) is swept over SWV. HS-DBP-1 specifically binds to PAE, knocking out the complementary DBP-1-10T-C-Fc or DBP-1-C-Fc, resulting in a decrease in the current signal (FIG. 8 a). Therefore, the change of the current can be monitored by sweeping square wave volt-ampere, and the DBP or other PAE can be quantitatively detected.
The experimental results show (fig. 8), that the screened DBP-1 can be used to construct electrochemical biosensor detection by engineering design, two different signal probe designs: DBP-1-10T-C-Fc (figure 8b) or DBP-1-C-Fc (figure 8C) realizes high-sensitivity detection on PAE, the detection limit is lower than 160ppt, and the detection interval is 160 ppt-1.6 ppm. Another 10. mu.M Hg2 +,Pb2+,Ni2+And a mixture of 10. mu.M kanamycin and 10. mu.M sulfadimethoxineThe response of 16 PAE mixed standard solutions with compounds (Kana + Sulf) all significantly less than 1.6ppm (about 4 μ M) demonstrated good selectivity of the electrochemical sensor (fig. 8 d).
Claims (9)
1. A method for screening and characterizing a single-stranded DNA aptamer of a phthalate plasticizer comprises the following steps: the method comprises the following steps: preparing a dibutyl phthalate derivative having an amino group at the terminal of one of linear fatty side chains by chemical synthesis; step two: covalently connecting a derivative of dibutyl phthalate with an amino group at the tail end of one linear fatty side chain to the agarose microspheres through a condensation reaction of the amino group and an ethylene oxide activated group on the agarose microspheres; step three: performing solid phase separation-based screening of nucleic acid aptamers; step four: the enriched library obtained was subjected to high throughput sequencing after screening.
2. The method of claim 1, wherein step one is as follows: (1) preparing mono-butyl phthalate; preparing DCC condensation products; preparation of dibutyl phthalate derivative in which one linear fatty side chain has amino group at terminal.
3. The process according to claim 2, characterized in that (1) the monobutyl phthalate is prepared as follows: placing 8.5g, 57mmol phthalic anhydride, 5ml n-butanol, 7.5 ml anhydrous treated tetrahydrofuran and clean dry magneton into 50 ml dry round bottom flask, heating and refluxing at 60 deg.C under magnetic stirring for 9 hr to obtain insoluble white solid, standing at room temperature, vacuum filtering, washing with tetrahydrofuran, vacuum filtering again, rotary steaming to obtain white slurry, adding CH2Cl2Then, white precipitate is formed, suction filtration and rotary evaporation are carried out to obtain oily liquid, 100 ml deionized water is added, extraction is carried out for 3 times by using ethyl acetate with the same volume, extraction is carried out for 1 time by using saturated NaCl, and finally anhydrous Na is used2SO4Drying the organic phase, standing overnight, filtering, spin-drying, separating and purifying with silica gel column, spin-drying, and vacuum-pumping to obtain 2.1g phthalic anhydrideMono-butyl acid, Agilent 1200 high performance liquid chromatography separation for unimodal, product purity; the DCC condensation product was prepared as follows: adding 1:1.2 of monobutyl phthalate and 5- (N-tert-butoxyamino) -1-pentanol into 20ml of anhydrous tetrahydrofuran, adding 1.158g of monobutyl phthalate into 20ml of anhydrous tetrahydrofuran, adding 5.495g of dicyclohexylcarbodiimide under magnetic stirring and ice-water bath, adding 0.217g of 4-dimethylaminopyridine after 10 minutes, adding 1.723g of 5- (N-tert-butoxyamino) -1-pentanol after 20 minutes, dissolving in 5ml of anhydrous tetrahydrofuran, dropwise adding, stirring for 1 hour in ice-water bath, removing the ice-water bath for reaction at room temperature for 27 hours, performing suction filtration on the reaction solution, washing a flask with anhydrous tetrahydrofuran, performing suction filtration, performing rotary evaporation on the filtrate, adding 20ml of petroleum ether for washing, performing suction filtration, placing in a refrigerator for overnight suction filtration, washing the flask with the petroleum ether for suction filtration, performing rotary evaporation, purifying the product through a silica gel column, the eluent is petroleum ether: ethyl acetate 8:2(v: v), rotary evaporating, purifying to obtain about 0.5g DCC condensation product, one-dimensional nuclear magnetic hydrogen spectrum characterization, VNMRS600 MHz, TMS as internal standard, CDCl3As a solvent, it was confirmed that the intended monobutyl phthalate was obtained; the dibutyl phthalate derivative in which one linear fatty side chain had an amino group at the terminal was prepared as follows: 0.5g DCC condensation product, 2ml dichloromethane and 2ml trifluoroacetic acid were added to a three-necked flask and magnetically stirred at room temperature for 40 minutes, then the mixture was filtered and rotary evaporated to give an oily liquid, which was extracted three times with ethyl acetate and saturated NaHCO3The reaction mixture was washed once, dried over anhydrous sodium sulfate, and finally filtered and rotary-evaporated to obtain 0.1g of a dibutyl phthalate derivative in which one of the linear aliphatic side chains had an amino group at the terminal.
4. The method of claim 1, wherein step two is as follows: 0.1g of epoxy-activated agarose microspheres was swollen with deionized water and washed repeatedly with 20mL of deionized water to obtain 350. mu.L of wet spheres. Then 0.2MNa pH 12 is used2CO3Washing the agarose beads, adding 46.7mg of a dibutyl phthalate derivative having an amino group at the end of one of the linear fatty side chains to 500. mu.L of the reaction systemThe organism is put under the condition of room temperature for oscillation reaction for 48 hours, and sodium acetate buffer solution with the composition of 0.1M sodium acetate, 0.5M NaCl and the pH value of 4.5 and NaHCO with the composition of 0.2M are used for reaction after the reaction is finished3Or Na2CO3And repeatedly and alternately washing the agarose microspheres for three times by using 0.5M NaCl pH 12 sodium carbonate buffer solution, finally washing by using water, metering the volume to 500 mu L, and storing in a refrigerator at 4 ℃ for later use.
5. The method of claim 1, wherein step three comprises 4 rounds of screening.
6. The method according to claim 5, characterized in that the random single-stranded DNA library Pool used is screened0 : TCCCACGCATTCTCCACATC(N40)CCTTTCTGTCCTTCCGTCACIs synthesized by Shanghai GmbH of biological engineering, purified by polyacrylamide gel electrophoresis PAGE, and is in the form of Pool0The full length is 80 bases, and the fixed sequence with the length of 20 bases at two ends and the random sequence with the length of 40 bases in the middle are included, and the fixed sequence is respectively connected with an upstream primer FP-SELEX: TCCCACGCATTCTCCACATC and downstream primer PO4-RP-SELEX:PO4GTGACGGAAGGACAGAAAGG, wherein the upstream primer and the downstream primer are synthesized by Takara Bio Inc., wherein the 5' end of the downstream primer is modified by phosphorylation, Pool0FP-SELEX and PO4-RP-SELEX is prepared into 100 mu M stock solution by using 1 xTris-EDTA buffer solution, and the stock solution is stored at the temperature of minus 80 ℃ for standby, wherein the 1 xTris-EDTA buffer solution is composed of 10mM Tris,1mM EDTA and pH8.0;
first screening: 1nmole of Pool0Diluting in 490. mu.L of binding buffer consisting of 20mM Tris, 100mM NaCl, 2mM MgCl, 5mM KCl, 1mM Ca chloride, 1% Tween20, 0.03% triton X-100, 2% DMSO, pH 7.9, heating the solution at 95 ℃ for 10 minutes, quenching in an ice-water bath for 5 minutes to room temperature, washing the prepared 100. mu.L agarose microspheres to which the derivative of dibutyl phthalate having an amino group at the terminal of one of the linear aliphatic side chains has been attached with the binding buffer three times, and then washing the microspheres three timesAdding the heated pool into the washed agarose microspheres0The solution is incubated at room temperature for 1 hour in a rotating way, a 10K ultrafiltration tube is used for separation, agarose beads are washed for three times by using a binding buffer solution, 100 mu L of the binding buffer solution is added, shaking and heating are carried out at 90 ℃ for ten minutes, then the ultrafiltration tube is used for separation, supernatant is collected, eluent is subjected to PCR amplification, the total volume of reaction is 2mL, the obtained PCR product is qualitatively characterized by PAGE, then the PCR product is purified by using an ethanol precipitation method, then the Lamda exonuclease digestion method is used for preparing single-stranded DNA, and the ethanol precipitation method is used for purifying to obtain the enriched single-stranded DNA library Pool1;
Rounds 2-4 SELEX: redissolving the Pool obtained by the first round of SELEX, quantifying, putting 200pmol for the next round of screening, wherein the specific screening process and conditions are the same as those of the first round of screening, and finally obtaining the enriched single-stranded DNA library Pool4。
7. The method of claim 6, wherein step four is as follows: and sending the obtained enrichment library to Beijing Nuo standing grain-derived bioinformatics science and technology Limited for high-throughput sequencing, wherein the high-throughput sequencing result shows that the first 100 single-stranded DNA sequences with the highest occurrence frequency are highly enriched and are rich in C basic groups.
8. The method of claim 7, wherein the single-stranded DNA sequence of step four is 5' CTTTCTGTCCTTCCGTCACN1-4TCCCACGCATTCTCCACAT3'。
9. The method of claim 8, wherein the single-stranded DNA sequence in step four is DBP-1: 5' CTTTCTGTCCTTCCGTCACATCCCACGCATTCTCCACAT3' or DBP-2:5' CTTTCTGTCCTTCCGTCACAGGTCCCACGCATTCTCCACAT3'。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610949635.5A CN106399317B (en) | 2016-10-26 | 2016-10-26 | Method for screening and characterizing single-stranded DNA aptamer of phthalate plasticizer |
CN201910033530.9A CN109609507B (en) | 2016-10-26 | 2016-10-26 | Single-stranded DNA aptamer of phthalate plasticizer, screening and characterization method of single-stranded DNA aptamer and electrochemical sensor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610949635.5A CN106399317B (en) | 2016-10-26 | 2016-10-26 | Method for screening and characterizing single-stranded DNA aptamer of phthalate plasticizer |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910033530.9A Division CN109609507B (en) | 2016-10-26 | 2016-10-26 | Single-stranded DNA aptamer of phthalate plasticizer, screening and characterization method of single-stranded DNA aptamer and electrochemical sensor |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106399317A CN106399317A (en) | 2017-02-15 |
CN106399317B true CN106399317B (en) | 2020-04-24 |
Family
ID=58014153
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610949635.5A Active CN106399317B (en) | 2016-10-26 | 2016-10-26 | Method for screening and characterizing single-stranded DNA aptamer of phthalate plasticizer |
CN201910033530.9A Active CN109609507B (en) | 2016-10-26 | 2016-10-26 | Single-stranded DNA aptamer of phthalate plasticizer, screening and characterization method of single-stranded DNA aptamer and electrochemical sensor |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910033530.9A Active CN109609507B (en) | 2016-10-26 | 2016-10-26 | Single-stranded DNA aptamer of phthalate plasticizer, screening and characterization method of single-stranded DNA aptamer and electrochemical sensor |
Country Status (1)
Country | Link |
---|---|
CN (2) | CN106399317B (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107446929B (en) * | 2017-08-31 | 2020-10-13 | 天津科技大学 | Aptamer for specifically recognizing ochratoxin A and preparation method thereof |
CN110261340A (en) * | 2019-05-24 | 2019-09-20 | 同济大学 | A kind of quick visualization analyzes the method and sensor of a variety of phthalate total amount of pollutant |
CN110257383B (en) * | 2019-06-24 | 2023-05-26 | 湖北师范大学 | Aptamer for specifically recognizing di (2-ethyl) hexyl phthalate as well as screening method and application thereof |
CN111135805B (en) * | 2020-01-21 | 2023-03-21 | 福州大学 | Aptamer functional extraction material for adsorbing phthalate, and preparation method and application thereof |
CN113249449B (en) * | 2021-06-28 | 2021-10-12 | 中国农业大学 | Method for determining Kd value based on real-time quantitative PCR of extension luminescent primer |
CN116515975A (en) * | 2023-04-21 | 2023-08-01 | 深圳市真迈生物科技有限公司 | Method for screening for aptamers using sequencing |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104655759A (en) * | 2015-02-13 | 2015-05-27 | 王利兵 | Method for detecting recovery rate of [2,2-bis(4-hydroxyphenol)propane] absorbed by using aptamer functional magnetic nanomaterial |
CN104749233A (en) * | 2015-03-31 | 2015-07-01 | 山东大学 | Bisphenol A sensor of nano-porous gold modified based on aptamer and method |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103487483B (en) * | 2013-09-12 | 2015-07-29 | 同济大学 | Electrochemical analysis method BDD electrode being constructed 17 beta estradiol aptamer sensor is modified based on dendritic gold |
-
2016
- 2016-10-26 CN CN201610949635.5A patent/CN106399317B/en active Active
- 2016-10-26 CN CN201910033530.9A patent/CN109609507B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104655759A (en) * | 2015-02-13 | 2015-05-27 | 王利兵 | Method for detecting recovery rate of [2,2-bis(4-hydroxyphenol)propane] absorbed by using aptamer functional magnetic nanomaterial |
CN104749233A (en) * | 2015-03-31 | 2015-07-01 | 山东大学 | Bisphenol A sensor of nano-porous gold modified based on aptamer and method |
Non-Patent Citations (4)
Title |
---|
A novel electrochemical aptasensor for bisphenol A assay basedon triple-signaling strategy;Peng Yu et al.;《Biosensors and Bioelectronics》;20151208;第22-28页 * |
Highly Sensitive Bisphenol-A Electrochemical Aptasensor Based on Poly(Pyrrole-Nitrilotriacetic Acid)-Aptamer Film;Imen Kazane et al.;《Anal. Chem.》;20160622;摘要,第7268页左栏至右栏,第7269页左栏第1-2、4段,图1-2 * |
邻苯二甲酸酯类增塑剂的检测标准现状分析;吴莉莉;《塑料工业》;20110430;第11-14页 * |
食品中邻苯二甲酸酯类增塑剂的检测方法进展;刘小文 等;《食品工业科技》;20141231;第35卷(第16期);第353-357页 * |
Also Published As
Publication number | Publication date |
---|---|
CN109609507B (en) | 2022-04-26 |
CN106399317A (en) | 2017-02-15 |
CN109609507A (en) | 2019-04-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106399317B (en) | Method for screening and characterizing single-stranded DNA aptamer of phthalate plasticizer | |
Yan et al. | Aptamer-based electrochemical biosensor for label-free voltammetric detection of thrombin and adenosine | |
CN101046461B (en) | Electrochemical sensor and its preparation process and use | |
Hua et al. | Label-free electrochemical cocaine aptasensor based on a target-inducing aptamer switching conformation | |
Zheng et al. | An electrochemical biosensor for the direct detection of oxytetracycline in mouse blood serum and urine | |
CN110004148B (en) | Alternariol aptamer affinity column and preparation method and application thereof | |
CN102288656A (en) | Sandwich-type electrochemical sensor for detecting ovarian SKOV-3 cancer cell | |
Xu et al. | Highly sensitive homogeneous electrochemiluminescence biosensor for microRNA-21 based on cascaded signal amplification of target-induced hybridization chain reaction and magnetic assisted enrichment | |
US10233442B2 (en) | Method for affinity purification | |
CN104777206A (en) | Aptamer electrode for detecting terramycin, and manufacturing method thereof | |
CN105126793B (en) | A kind of preparation method based on single stranded DNA nucleic acid aptamers modification hybrid quartz capillary integral post | |
Liu et al. | A simple regenerable electrochemical aptasensor for the parallel and continuous detection of biomarkers | |
CN103901020A (en) | Method for detecting DNA (Deoxyribose Nucleic Acid) transmethylase by using electrochemical luminescence biosensor and application of biosensor | |
Li et al. | An aptasensor for the detection of ampicillin in milk using a personal glucose meter | |
Dun-Ming et al. | Application of aptamers in food safety | |
CN102784628B (en) | Solid-phase micro extraction fiber extraction head and preparation method thereof | |
CN109001164B (en) | Photoelectric biosensor based on manganoporphyrin quenched CdSe quantum dots and preparation method and application thereof | |
KR100930974B1 (en) | DNA aptamer binding to Retinol Binding Protein 4 with specificity and production method thereof | |
KR101822881B1 (en) | Aptamer specifically binding to nonylphenol and detecting method using thereof | |
KR101678946B1 (en) | Nucleic Acid Aptamer Capable of Specifically Binding to 25-hydroxy Vitamin D3 and Uses Thereof | |
KR101086026B1 (en) | DNA aptamers binding to visfatin with specificity and production method thereof | |
CN112094416A (en) | Lanthanide coordination polymer fluorescent probe for fluorescence detection of sialic acid in blood plasma, preparation method and detection method | |
CN114606294B (en) | Curve establishment method, detection method and kit for nucleic acid detection | |
CN104152455B (en) | The aptamers electrochemica biological sensor of gram rumba amine aptamers and detection Ke Lunba amine | |
CN113980970B (en) | Aptamer specifically binding with sea anemone cytolysin GT-4 and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |