CN114561446B - Universal aptamer biosensor and application thereof in field of marker detection - Google Patents
Universal aptamer biosensor and application thereof in field of marker detection Download PDFInfo
- Publication number
- CN114561446B CN114561446B CN202210198403.6A CN202210198403A CN114561446B CN 114561446 B CN114561446 B CN 114561446B CN 202210198403 A CN202210198403 A CN 202210198403A CN 114561446 B CN114561446 B CN 114561446B
- Authority
- CN
- China
- Prior art keywords
- aptamer
- detection
- atp
- probe
- dna
- 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
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 246
- 238000001514 detection method Methods 0.000 title claims abstract description 117
- 239000003550 marker Substances 0.000 title claims abstract description 17
- 239000000523 sample Substances 0.000 claims abstract description 104
- 230000003321 amplification Effects 0.000 claims abstract description 44
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 44
- 239000011324 bead Substances 0.000 claims abstract description 15
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 9
- 230000000692 anti-sense effect Effects 0.000 claims abstract description 5
- 108090000190 Thrombin Proteins 0.000 claims description 59
- 229960004072 thrombin Drugs 0.000 claims description 59
- VYXSBFYARXAAKO-UHFFFAOYSA-N ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate;hydron;chloride Chemical compound [Cl-].C1=2C=C(C)C(NCC)=CC=2OC2=CC(=[NH+]CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-UHFFFAOYSA-N 0.000 claims description 42
- 238000006243 chemical reaction Methods 0.000 claims description 31
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 30
- 229960002685 biotin Drugs 0.000 claims description 20
- 239000011616 biotin Substances 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 20
- 235000020958 biotin Nutrition 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 14
- 238000004873 anchoring Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 239000005543 nano-size silicon particle Substances 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 10
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 108010090804 Streptavidin Proteins 0.000 claims description 9
- 230000004048 modification Effects 0.000 claims description 9
- 238000012986 modification Methods 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 8
- 230000000903 blocking effect Effects 0.000 claims description 7
- 230000000295 complement effect Effects 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 150000001408 amides Chemical class 0.000 claims description 5
- 239000002585 base Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000009833 condensation Methods 0.000 claims description 4
- 230000005494 condensation Effects 0.000 claims description 4
- 239000012502 diagnostic product Substances 0.000 claims description 4
- 229940014800 succinic anhydride Drugs 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 3
- 238000009007 Diagnostic Kit Methods 0.000 claims description 2
- 239000006087 Silane Coupling Agent Substances 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 229940043267 rhodamine b Drugs 0.000 claims description 2
- 239000011856 silicon-based particle Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- ODHXBMXNKOYIBV-UHFFFAOYSA-N triphenylamine Chemical compound C1=CC=CC=C1N(C=1C=CC=CC=1)C1=CC=CC=C1 ODHXBMXNKOYIBV-UHFFFAOYSA-N 0.000 claims description 2
- WJQDJDVDXAAXSB-UHFFFAOYSA-N 5-sulfanylidenepyrrolidin-2-one Chemical compound O=C1CCC(=S)N1 WJQDJDVDXAAXSB-UHFFFAOYSA-N 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 27
- 230000035945 sensitivity Effects 0.000 abstract description 19
- 238000011896 sensitive detection Methods 0.000 abstract description 8
- 238000012795 verification Methods 0.000 abstract description 5
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 122
- 108010081589 Becaplermin Proteins 0.000 description 65
- 108020004414 DNA Proteins 0.000 description 59
- 238000009739 binding Methods 0.000 description 31
- 230000027455 binding Effects 0.000 description 27
- 201000011510 cancer Diseases 0.000 description 24
- 238000013461 design Methods 0.000 description 23
- 210000002966 serum Anatomy 0.000 description 21
- 150000007523 nucleic acids Chemical class 0.000 description 18
- 239000000872 buffer Substances 0.000 description 16
- 238000002189 fluorescence spectrum Methods 0.000 description 15
- 238000011084 recovery Methods 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 238000001962 electrophoresis Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 11
- 108020004707 nucleic acids Proteins 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 230000009471 action Effects 0.000 description 9
- 230000003993 interaction Effects 0.000 description 9
- 238000007885 magnetic separation Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 238000003917 TEM image Methods 0.000 description 7
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 7
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 239000011734 sodium Substances 0.000 description 7
- 239000007984 Tris EDTA buffer Substances 0.000 description 6
- 239000012472 biological sample Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- VYXSBFYARXAAKO-WTKGSRSZSA-N chembl402140 Chemical compound Cl.C1=2C=C(C)C(NCC)=CC=2OC2=C\C(=N/CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-WTKGSRSZSA-N 0.000 description 6
- 239000011148 porous material Substances 0.000 description 6
- 239000011535 reaction buffer Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- XJZNCUDTWKPVBJ-UHFFFAOYSA-N 3-triethylsilylpropan-1-amine Chemical compound CC[Si](CC)(CC)CCCN XJZNCUDTWKPVBJ-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000000171 quenching effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000012496 blank sample Substances 0.000 description 3
- 230000009137 competitive binding Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000003795 desorption Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000001960 triggered effect Effects 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 239000000439 tumor marker Substances 0.000 description 3
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 2
- 238000007702 DNA assembly Methods 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000021523 carboxylation Effects 0.000 description 2
- 238000006473 carboxylation reaction Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 230000005281 excited state Effects 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000005283 ground state Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000012798 spherical particle Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 125000001834 xanthenyl group Chemical group C1=CC=CC=2OC3=CC=CC=C3C(C12)* 0.000 description 2
- -1 15 mL) Chemical compound 0.000 description 1
- BLSAPDZWVFWUTL-UHFFFAOYSA-N 2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound OS(=O)(=O)C1CC(=O)NC1=O BLSAPDZWVFWUTL-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000008836 DNA modification Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108091027757 Deoxyribozyme Proteins 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
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 238000000696 nitrogen adsorption--desorption isotherm Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 210000004915 pus Anatomy 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000015607 signal release Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 1
- IARLBMZSSJJOCU-UHFFFAOYSA-N sodium;1-oxidopyrrolidine-2,5-dione Chemical compound [Na+].[O-]N1C(=O)CCC1=O IARLBMZSSJJOCU-UHFFFAOYSA-N 0.000 description 1
- 238000002336 sorption--desorption measurement Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- GOMLCFUVZKLQCO-HTLAMOOLSA-N thrombin aptamer Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2CO)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)[C@@H](OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)O)C1 GOMLCFUVZKLQCO-HTLAMOOLSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/49—Platelet-derived growth factor [PDGF]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/974—Thrombin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nanotechnology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a universal aptamer biosensor and application thereof in the field of marker detection. Existing biosensing typically requires designing the identification unit of the entire sensor for the target object, significantly limiting the flexibility of sensor application. The invention aims to provide a general biosensor, which can realize sensitive detection by replacing an identification element. In order to achieve the above purpose, the universal aptamer biosensor provided by the invention comprises a magnetic probe module and a signal amplification module, wherein the magnetic probe module is a magnetic bead for modifying an identification probe, and the identification probe is provided with an aptamer sequence of a detection target; the signal amplification module comprises a porous carrier, wherein the porous carrier is used for encapsulating fluorescent dye, and the surface of the porous carrier is modified with a connecting chain, and the connecting chain comprises an antisense sequence of an identification probe or an aptamer sequence. Proved by verification, the sensor can be applied to detection of various tumor markers and has good detection sensitivity.
Description
Technical Field
The invention belongs to the technical field of a marker detection biosensor, and particularly relates to a universal aptamer biosensor, application of the universal aptamer biosensor in the field of marker detection and a detection method of a marker.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
As a material basis for vital activities, the homeostasis of biomolecules in living organisms plays an indispensable role in various cellular processes such as cell proliferation, differentiation and apoptosis. Among these, abnormal expression of certain biomolecules, which are called cancer markers, leads to the occurrence and development of cancer. For example, adenosine triphosphate (Adenosine triphosphate, ATP), as a basic energy molecule for cellular activity, is expressed at significantly higher levels in the cancer cell microenvironment than in normal cells; thrombin, a protease that catalyzes the conversion of fibrinogen to fibrin, is expressed at elevated levels in leukemia cells; platelet derived growth factor-BB (Platelet-derived growth factor BB, PDGF-BB), an important cytokine, is overexpressed in a variety of cancer cells. The occurrence of abnormal expression of cancer markers is often earlier than the clinical symptoms of cancer, and their expression level has become an important basis for early diagnosis of cancer. Notably, most cancer markers are only expressed in trace amounts, and the abnormal changes at the early stages of cancer are extremely weak. Therefore, the development of a sensitive detection method capable of distinguishing a weak change of a cancer marker is of great importance for early detection and in-depth research of cancer.
Fluorescent sensors have been attracting attention in sensitive detection of cancer markers due to their strong signal response. In particular, some fluorescent sensors, by combining with nucleic acid amplification strategies, exhibit greater sensitivity in the detection of cancer markers. Currently, most fluorescent sensors use protease-assisted DNA synthesis/hydrolysis or enzyme-free DNA assembly to obtain amplified signals. However, the non-specific action of proteases and unavoidable leakage in DNA assembly can lead to spurious signals, somewhat reducing the accuracy of the detection. Furthermore, in most reported fluorescence sensors, the sequence of recognition units often participates in the signal amplification process, so the entire sensor has to be designed for each specific target, which limits the flexibility of sensor detection. Therefore, in order to solve the above-described problems, it is necessary to develop a general-purpose sensor having a novel amplification mode for sensitive detection of various targets.
Disclosure of Invention
In order to realize sensitive detection of the micro-markers, the design combines magnetic separation and clustered fluorescent amplification, and designs a modularized universal aptamer sensor which is a universal detection platform, and the probe is only required to be identified according to a detection target design, so that 'plug and play' can be realized, and the application is wider.
The universal aptamer sensor provided by the invention consists of a magnetic probe module and a cluster amplification module. The Magnetic probe module is constructed by attaching a recognition probe composed of an aptamer and an anchor DNA to a Magnetic Bead (MBD). The structural basis of the clustered fluorescent amplifying module is mesoporous silicon nanoparticles (Mesoporous silica nanoparticle, MSN) which are constructed by loading Rhodamine 6G (Rhodamine 6G, rh6G) in cavities thereof and modifying connecting chains (anchoring antisense sequences of DNA) on surfaces thereof through amide condensation. The ligation strand is responsible for blocking the dye in the MSN and binding the exposed anchored DNA. Competitive binding of the target to the aptamer exposes the anchor DNA when the target is present. The exposed anchored DNA captures the clustered fluorescent amplification module by hybridization to the ligation strand. Under the action of an externally applied magnetic field, the captured clustered fluorescent amplifying module is separated along with the movement of the MBD, rh6G in the clustered fluorescent amplifying module is released by heating, and fluorescence is recovered to indicate the concentration of a target object. In the method, an uncaptured cluster fluorescent amplifying module can be removed through magnetic separation, so that the background is lower when the aptamer sensor is used for detecting cancer markers; due to the design of clustered fluorescent amplification, a single target binding event is converted into a cluster of Rh6G signals, so that amplified signals can be generated, and the detection sensitivity is improved; thanks to its modular design, the aptamer sensor can be easily extended to the detection of multiple cancer markers by simply replacing the recognition probes by "plug and play". After feasibility verification and condition optimization, the aptamer sensor is successfully used for sensitively detecting ATP, thrombin and PDGF-BB in a buffer system, the detection limits are respectively 2.1nM, 4.1pM and 2.4pM, and three targets are successfully detected in a human serum system. The invention discloses a universal aptamer sensing method for sensitively detecting cancer markers, which can provide an effective tool for early diagnosis of cancers.
Based on the research results, the invention provides the following technical scheme:
in a first aspect of the present invention, there is provided a universal aptamer biosensor, the biosensor comprising a magnetic probe module and a signal amplification module; the magnetic probe module is a magnetic bead for modifying an identification probe, and the identification probe is provided with an aptamer sequence of a detection target;
the signal amplification module comprises a porous carrier, wherein the porous carrier is used for encapsulating fluorescent dye, and the surface of the porous carrier is modified with a connecting chain, and the connecting chain comprises an antisense sequence of an identification probe or an aptamer sequence.
In the magnetic probe module according to the first aspect, the:
Preferably, the surface of the magnetic bead is modified with streptavidin, and the recognition probe is connected to the surface of the magnetic bead by means of the specific combination of the streptavidin and the biotin.
In the signal amplifying module according to the first aspect, the signal amplifying module includes:
The porous carrier is a spherical material with hollow inside and through holes distributed on the surface of the carrier, the diameter is 1-2 mu m, the aperture is 1-5 nm, and the surface can be fixedly connected with a chain in a physical and chemical mode; the carrier capable of meeting the structural characteristics can be applied to the aptamer biosensor provided by the invention. In one embodiment of the present invention, the spherical carrier is a mesoporous silica nano carrier.
Preferably, the porous carrier is an aminated mesoporous silicon nanoparticle, and is connected with the connecting chain through an amide bond.
Further, the mesoporous silicon nanoparticle adopts a copolycondensation method, and the preparation method comprises the following steps: slowly adding hexadecyl trimethyl ammonium bromide (CTAB) aqueous solution into alkali liquor, stirring and heating for reacting for a period of time, dropwise adding Tetraethoxysilane (TEOS) and a silane coupling agent (specifically, APTES) into the reaction solution, and continuously heating for reacting to obtain a white solid; separating the white solid, washing and drying, adding hydrochloric acid and methanol solution, and refluxing to remove CTAB, thus obtaining MSN-NH 2.
Preferably, the porous carrier internally encapsulates a fluorescent dye, wherein the fluorescent dye is one of rhodamine 6G, rhodamine B, triphenylamine and derivatives thereof. Based on the design thought of the invention, the aptamer biosensor preferably does not express a fluorescent signal before magnetic separation, and when the aptamer is combined with a detection target, the anchored DNA is combined with a connecting chain, and the fluorescent dye blocked in the carrier is released to express the fluorescent signal. In order to meet the above-mentioned assumption, in one embodiment provided by the present invention, rhodamine 6G is adopted as the fluorescent dye, rhodamine 6G is loaded into the pore cavity of the carrier, rh6G molecules in the pore aggregation state are close to each other, causing dipole superposition of xanthene ring structures between adjacent Rh6G molecules, and molecules in the excited state are attenuated or relaxed through a non-radiative channel to return to the ground state, resulting in fluorescence quenching (aggregation-induced quenching effect, ACQ effect). After the aptamer is combined with a detection target, rh6G loses the blocking of a connecting chain and is released from a carrier, the released Rh6G is far away from each other, the ACQ effect disappears, and fluorescence is recovered, so that amplification of a detection signal is realized.
Furthermore, the mesoporous silicon nanoparticle surface is modified by connecting chains through amide bonds; in one embodiment, the connection means is as follows: carboxylation is carried out on the MSN-NH 2, and a connecting chain is modified to the surface of the mesoporous silicon particle through amide condensation.
A specific preparation method of the signal amplification module is as follows: adding succinic anhydride into a dimethylformamide solution of MSN-NH 2 to react to obtain the mesoporous silicon nanoparticles (MSN-COOH) with carboxylated surfaces; adding the suspension of MSN-COOH into the Rh6G solution, stirring in a dark place to obtain MSN-COOH red solid (Rh6G@MSN-COOH) carrying Rh6G, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and sulfosuccinimide (Sulfo-NHS) into the suspension of Rh6G@MSN-COOH, stirring for reaction to activate carboxyl, adding PBS buffer solution and an aminated connecting chain, and reacting in a dark place to obtain the signal amplification module.
In one embodiment of the universal aptamer biosensor according to the first aspect, the recognition probe comprises an anchor DNA and an aptamer, wherein:
The 3' end of the aptamer is provided with an aptamer sequence for combining with a detection target; the appropriate length of the aptamer sequence is 20-40 bases, preferably 25-40 bases; the 5 'end of the anchoring DNA is provided with a complementary sequence of a connecting chain, the 3' end of the anchoring DNA is modified with biotin, and the anchoring DNA is connected to the surface of the magnetic bead through the specific combination of the biotin and streptavidin; the 5 'end of the connecting strand has a complementary sequence of anchored DNA, the 3' end has amino (-NH 2) group connected to the surface of the porous carrier through an amide bond, and the rest sites are blocked through a blocking strand.
In one example of the above embodiment, a nucleic acid sequence combination for ATP detection is provided, the specific sequences are as follows:
An aptamer: GAGAGAACCTGGGGGAGTATTGCGGAGGAAGGT (SEQ ID NO. 1);
anchoring the DNA: CCCAGGTTCTCTCTCACACATCTGT-biotin (SEQ ID NO. 2);
connecting chain: GAGAGAACCTGGGGATATGCTAGACTATCG-NH 2 (SEQ ID NO. 3);
closed chain: CGATAGTCTAGCATA (SEQ ID NO. 4).
In yet another example, a combination of nucleic acid sequences for thrombin detection is provided, the specific sequences being as follows;
an aptamer: GCTCGGAGTCCGTGGTAGGGCAGGTTGGGGTGACT (SEQ ID NO. 5);
Anchoring the DNA: ACGGACTCCGAGCTCACACATCTGT-biotin (SEQ ID NO. 6);
Connecting chain: GCTCGGAGTCCGTGATATGCTAGACTATCG-NH 2 (SEQ ID NO. 7);
Closed chain: CGATAGTCTAGCATA (SEQ ID NO. 8).
In yet another embodiment of the universal aptamer biosensor, when the target to be detected has a double aptamer binding site, the 5 'end of the recognition probe has an aptamer sequence, the 3' end of which modifies biotin, and is linked to the surface of the magnetic bead through the specific binding of biotin and streptavidin; the 5 'end of the connecting chain has the same aptamer sequence, the 3' end has amino (-NH 2) and is connected to the surface of the porous carrier through an amide bond, and the rest sites are blocked through a blocking chain.
In one example of the above embodiment, a combination of nucleic acid sequences for PDGF-BB detection is provided, with the following specific sequences:
anchoring the DNA:
CAGGCTACGGCACGTAGAGCATCACCATGATCCTGTCACACATCTGT-biotin(SEQ ID NO.9)；
Ligation of DNA:
CAGGCTACGGCACGTAGAGCATCACCATGATCCTGGATCTACTAGACTATCG-NH2(SEQ ID NO.10)；
Closed chain: CGATAGTCTAGC (SEQ ID NO. 11).
In a second aspect, the invention provides an application of the universal aptamer biosensor in the field of marker detection.
Applications of the second aspect include, but are not limited to, any of the following:
(1) For preparing diagnostic products;
(2) For the prevention, diagnosis or assessment of prognostic conditions;
(3) For screening of active compounds.
In the application of the above aspect (1), the diagnostic product includes, but is not limited to, diagnostic kits, diagnostic chips and other possible diagnostic systems.
In the application of the above (2) and (3), the marker is a marker in an in vitro test sample, and the in vitro test sample includes a clinical biological sample, and specific examples include one or more of serum (plasma), whole blood, secretion, cotton swab, pus, body fluid, tissue, organ, paraffin section, and the like.
Further, the marker is a tumor diagnosis or prognosis marker; in specific examples, such as ATP, thrombin or PDGF-BB.
In a third aspect of the present invention, there is provided a method of detecting a marker, the method comprising the steps of: when the universal aptamer biosensor in the first aspect is added into a sample to be detected and combined for a certain period, the universal aptamer biosensor combined with the target object is moved out through an external magnetic field, and is heated to promote release of Rh6G, and the dosage of the target object is detected by detecting the fluorescence intensity of Rh 6G.
Preferably, the binding time between the universal aptamer biosensor and the target is 30-50 min.
Preferably, the heating temperature is 75-85 ℃.
The beneficial effects of the above technical scheme are:
In one embodiment of the invention, a modularized universal aptamer sensor is developed, and the application of the aptamer sensor in ATP, thrombin and PDGF-BB detection is examined, wherein the adaptive sensor has the following advantages:
1. thanks to the design of magnetic separation and the design of clustered signal amplification, the aptamer sensor converts a target binding event into a cluster of fluorescent signal release events, and can conveniently remove an uncaptured signal amplification module, so that the aptamer sensor has higher sensitivity in ATP, thrombin and PDGF-BB detection, and detection limits are respectively 2.1nM, 4.1pM and 2.4pM;
2. Thanks to the modular design and the "plug and play" assembly mode, the versatility of the aptamer sensor is improved, and the aptamer sensor can be easily extended to the detection of other cancer markers by simply replacing the corresponding recognition probes;
3. By combining the magnetic separation technology, the modularized design and the cluster signal amplification strategy, the nano sensor provides a new thought for the design of a general aptamer sensor, and has important significance for the flexible, specific and sensitive detection of cancer markers.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of a universal aptamer sensor of the invention for detection of a tag.
FIG. 2 is a structural representation of MSN-NH 2 in example 1;
Wherein fig. 2 is (a) a TEM image; FIG. 2 (B) is a nitrogen adsorption/desorption isotherm; FIG. 2 (C) is a pore size distribution curve; FIG. 2 (D) is a TEM image of MBD; FIG. 2 (E) is a TEM image of MBD@MSN-LINKING STRAND; FIG. 2 (F) shows the Zeta potential of MSN-NH 2, MSN-COOH, MSN-LINKING STRAND, MBD, MBD@MSN-LINKING STRAND.
FIG. 3 shows the results of polyacrylamide gel electrophoresis (PAGE) verification of ATP detection as described in example 2;
FIG. 3 (A) shows the feasibility of nucleic acid strand interactions for ATP detection for non-denaturing PAGE; lane M: marker, lane 1: aptamer (500 nM), lane 2: anchored DNA (500 nM), lane 3: recognition probe (500 nM), lane 4: connecting strand (500 nM), lane 5: ligation strand (500 nM) +Anchor DNA (500 nM), lane 6: recognition probe (500 nM) + ligation strand (500 nM), lane 7: recognition probe (500 nM) +ligation strand (500 nM) +ATP (5.0. Mu.M);
FIG. 3 (B) is a fluorescence emission spectrum of an aptamer sensor under different conditions.
FIG. 4 is a graph showing the results of optimizing parameters of the ATP detecting aptamer sensor described in example 1;
Wherein, FIG. 4 (A) is Mg 2+ concentration; FIG. 4 (B) is the ATP binding time; FIG. 4 (C) is the release temperature; FIG. 4 (D) is the DNA ligation time; FIG. 4 (E) is Na + concentration; FIG. 4 (F) is the effect of Rh6G@MSN-LINKING STRAND concentration on DNAzyme walker fluorescence signal intensity; histograms represent the fluorescence signal intensity of the aptamer sensor in the presence (positive group, F) and in the absence (negative group, F 0) of ATP under different reaction conditions; the line graph represents the ratio of F/F 0 under different reaction conditions.
FIG. 5 shows the detection sensitivity of the ATP detecting aptamer sensor described in example 1;
Wherein, FIG. 5 (A) is the fluorescence emission spectrum of the aptamer sensor at different concentrations of ATP; the ATP concentrations corresponding to curves a to p are 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 1.0, 2.0, 3.0, 5.0, 10, 20, 50 μm, respectively;
FIG. 5 (B) is a graph showing the net fluorescence signal intensity F-F 0 as a function of ATP concentration for an aptamer sensor for ATP detection; the inset shows the linear relationship between F-F 0 and ATP concentration; error bars are standard deviations of three parallel experiments.
FIG. 6 shows fluorescence signal intensities of aptamer sensors with different targets as described in example 1;
Wherein, the ATP concentration is 20 mu M; TTP, CTP, GTP, dATP was 100 μm; error bars are standard deviations of three parallel experiments.
FIG. 7 is a comparison of fluorescence signal intensities of the aptamer sensor described in example 1 for detecting ATP in a reaction buffer system and a human serum system;
error bars are standard deviations of three parallel experiments.
FIG. 8 is a schematic diagram of the thrombin detection aptamer sensor described in example 2;
Wherein, FIG. 8 (A) magnetic separation and clustered fluorescence amplification combined modular universal aptamer sensor for thrombin detection principle; (B) Non-denaturing PAGE verifies the feasibility of aptamer sensors for thrombin detection; lane M: marker, lane 1: aptamer (500 nM), lane 2: anchored DNA (500 nM), lane 3: recognition probe (500 nM), lane 4: connecting strand (500 nM), lane 5: recognition probe (500 nM) + anchor DNA (500 nM), lane 6: recognition probe (500 nM) + ligation strand (500 nM), lane 7: recognition probe (500 nM) +ligation strand (500 nM) +thrombin (1.0. Mu.M); (C) Fluorescence emission spectra of aptamer sensors under different conditions.
FIG. 9 is a schematic diagram of the PDGF-BB detection aptamer sensor described in example 2;
Wherein FIG. 9 (A) verifies the feasibility of aptamer sensors for PDGF-BB detection for non-denaturing PAGE; lane M: marker, lane 1: aptamer probe 1 (500 nM), lane 2: aptamer probe 2 (500 nM), lane 3: aptamer probe 1 (500 nM) +PDGF-BB (1.0. Mu.M), lane 4: aptamer probe 2 (500 nM) +PDGF-BB (1.0. Mu.M), lane 5: aptamer probe 1 (500 nM) +aptamer probe 2 (500 nM), lane 6: aptamer probe 1 (500 nM) +aptamer probe 2 (500 nM) +PDGF-BB (1.0. Mu.M); panel (B) shows fluorescence emission spectra of aptamer sensors under different conditions.
FIG. 10 is a plot of the sensitivity of the aptamer sensor described in example 2 to detect thrombin;
Wherein, FIG. 10 (A) is the fluorescence emission spectrum of the sensor at different concentrations of thrombin; the thrombin concentrations corresponding to curves a to o are 0.02, 0.04, 0.08, 0.2, 0.4, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 20 nM, respectively; FIG. 10 (B) is a graph showing the net fluorescence signal intensity F-F 0 as a function of thrombin concentration for thrombin detection by the sensor; the inset shows the linear relationship of F-F 0 and thrombin concentration; error bars are standard deviations of three parallel experiments.
FIG. 11 is a plot of the sensitivity results of the aptamer sensor described in example 2 for detecting PDGF-BB;
FIG. 11 (A) shows fluorescence emission spectra of aptamer sensors at different concentrations of PDGF-BB; the PDGF-BB concentrations corresponding to curves a to o are 0.02, 0.04, 0.08, 0.2, 0.4, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 20nM, respectively; (B) The aptamer sensor is used for detecting a change curve of fluorescence signal intensity F-F 0 along with the concentration of PDGF-BB when the PDGF-BB is detected; the inset shows the linear relationship between F-F 0 and PDGF-BB concentrations; error bars are standard deviations from three parallel experiments.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
As described in the background, sensitive detection of cancer markers is of great importance for early detection and in-depth investigation of cancer, and fluorescence sensors combined with nucleic acid amplification strategies show higher sensitivity in cancer marker detection, but the non-specific action of proteases in conventional fluorescence sensors may lead to false positive signals. In order to solve the technical problems, the invention provides a modularized universal aptamer sensor with magnetic separation and cluster fluorescent amplification.
The principle of the magnetic separation combined with clustered fluorescent amplified modular universal aptamer sensor for sensitive detection of cancer markers is shown in fig. 1. Firstly, a magnetic probe module is prepared by modifying streptavidin-modified magnetic beads (MBD-SA) with a recognition probe (composed of an aptamer and an anchor DNA) by utilizing the specific binding action of streptavidin and biotin. Then, rh6G is loaded into the pore space of the MSN, and as Rh6G molecules in the in-pore aggregation state are close to each other, causing dipoles of the xanthene ring structure between adjacent Rh6G molecules to overlap, molecules in the excited state are attenuated or relaxed back to the ground state through a non-radiative channel, resulting in fluorescence quenching (aggregation-induced quenching effect, ACQ effect). Next, a binding chain (LINKING STRAND) was modified on the MSN surface by amide condensation to prepare a clustered fluorescent amplification module. The ligation strand contains an antisense sequence of the anchor DNA responsible for binding the magnetic probe module and blocking the MSN. The following will explain the principle by selecting ATP as a model. When ATP is present, a competitive binding reaction occurs between ATP and the aptamer, exposing the anchored DNA, and the MBD captures the clustered fluorescent amplification module by hybridization of the surface anchored DNA to the ligation strand of the clustered fluorescent amplification module. Under the action of an external magnetic field, the MBD and the clustered fluorescent amplifying module captured by the MBD are separated, and the blocking of the MSN is released by heating, so that Rh6G is released. The released Rh6G was far away from each other, the ACQ effect disappeared, and fluorescence was restored for indicating the ATP concentration. Thanks to its modular design, the sensor can be used in the detection of other cancer markers (thrombin and PDGF-BB) by simply replacing the recognition probes "plug and play".
Reagents and apparatus
The nucleic acids used in this example (see Table 1-3.3 for specific sequences) were purchased from Biotechnology Inc. (Shanghai, china). Streptavidin-modified magnetic beads (MBD-SA) were purchased from Chinesemedicine Biotechnology Co., ltd., shanghai, china. Tetraethoxysilane (TEOS) is purchased from national drug group limited (shanghai, china). Cetyl trimethylammonium bromide (CTAB), rhodamine 6G (Rh 6G) and succinic anhydride were purchased from Soy Biotech Co., ltd (Beijing, china). 3-aminopropyl triethylsilane (APTES) was purchased from Michelin Biochemical technologies Co., ltd (Beijing, china). 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Source leaf Biotechnology Co., ltd (Shanghai, china). ATP, TTP, CTP, GTP, dATP and N-hydroxysuccinimide sodium salt were purchased from BBI life sciences Co., ltd (Shanghai, china). Thrombin and platelet-derived growth factor BB (PDGF-BB) were purchased from beijing NEB limited (beijing, china). The chemical reagents used in this example were all analytically pure, and the solutions and buffers used were all prepared using ultrapure water.
TABLE 1 nucleic acid sequences for ATP detection
Note that: the italic portion of the Aptamer chain represents the Aptamer sequence of ATP; the underlined parts of the anchor DNA and the ligation strand in the table represent the complements of the two, biotin in the anchor DNA represents biotin modification, and NH 2 in the ligation strand represents amino modification; the italic portions of the connecting strand and the closed strand represent the complementary sequences of both.
TABLE 2 nucleic acid sequences for thrombin detection
Note that: the italic portion of the Aptamer chain represents the Aptamer sequence of ATP; the underlined parts of the anchor DNA and the ligation strand in the table represent the complements of the two, biotin in the anchor DNA represents biotin modification, and NH 2 in the ligation strand represents amino modification; the italic portions of the connecting strand and the closed strand represent the complementary sequences of both.
TABLE 3 nucleic acid sequences for PDGF-BB detection
Note that: underlined parts in the aptamer probe 1 and the aptamer probe 2 represent aptamer sequences, biotin in the aptamer probe 1 represents biotin modification, and NH 2 in the aptamer probe 2 represents amino modification; the italics in the closed strand and aptamer probe 2 represent the complementary sequences of both.
Polyacrylamide gel electrophoresis (PAGE) gels were imaged using a Bio-RAD GelDocTM XR + gel imaging system (USA). Transmission Electron Microscope (TEM) images were taken using a JEM 2100 electron microscope (japan). ASAP 2020 multi-station expansion specific surface area and porosity Analyzer (USA) was used to plot Brunauer-Emmett-Teller (BET) nitrogen adsorption-desorption isotherms. Dynamic Light Scattering (DLS) and surface Zeta potential data of the nanoparticles were measured using a Malvern nanodynamics analyzer (uk). FT-IR spectra were recorded using a Tensor II spectrometer (Germany). Fluorescence signal intensity was measured and fluorescence emission spectra were recorded using a Hitachi F-7000 fluorescence spectrophotometer (Japan). Absorbance was measured and ultraviolet-visible spectrum was recorded using a hitachi U-2910 spectrometer (japan).
Gel electrophoresis experiments
The feasibility of designed nucleic acid strand interactions was verified using non-denaturing PAGE. First, a 12% PAGE gel was prepared, and an acrylamide solution (7.5 mL, 40%), a 5 XTBE buffer (Tris 88mM, boric acid 88mM,EDTA 2.0 mM,pH 8.3,5.0mL), N, N, N ', N' -tetramethyl ethylenediamine (18. Mu.L), an ammonium persulfate solution (0.1 g mL -1, 180. Mu.L) and ultrapure water (12.5 mL) were mixed uniformly to prepare a gel. Samples were injected into the gel and then electrophoresed. The electrophoresis conditions were 8℃and 25mA (constant flow mode), 50min (electrophoresis time), and the electrophoresis buffer was 1 XTBE buffer. After electrophoresis, the gel was stained in a SYBR Gold solution in the dark for 40min, and the stained gel was imaged in an imager.
Construction of magnetic probe modules
MBD-SA (10 mg mL -1, 2.0. Mu.L) was resuspended in PBS buffer after washing and activation 5 times (15 min each) with TTL buffer (Tris 100mM, tween-20.1%, liCl 1.0M, pH 8.0, 200. Mu.L). Biotin-modified DNA (recognition probe, 2.0. Mu.M, 20. Mu.L) was then added to the MBD-SA suspension and dissolved in PBS buffer. After shaking for 2 hours at room temperature, the magnetic beads (MBD-recognition probe) modified with the recognition probes were separated from the reaction system by an external magnetic field. After washing with PBS, MBD-recognition probe was resuspended in PBSM(10 mM NaH2PO4,10mM Na2HPO4,137mM NaCl,2.7mM KCl,MgCl2 6.0mM,40.0μL) buffer. The washing process is completed through the action of an externally applied magnetic field.
Synthesis of aminated mesoporous silicon nanoparticles
The aminated mesoporous silicon nano particles (MSN-NH 2) are synthesized by a copolycondensation method. Specifically, CTAB (0.25 g) was dissolved in deionized water (120 mL) and NaOH solution (2.0M, 0.9 mL) was slowly added with stirring. After stirring 5min, the reaction was heated to 80 ℃. After the reaction was continued for 30 minutes, TEOS (1.2 mL) and APTES (0.25 mL) were added dropwise to the reaction solution. The mixture was kept at 80℃for a further 2h to give a white solid. After stopping the reaction and cooling to room temperature, the solids were separated by centrifugation. The separated solids were washed 3 times with ethanol and water, respectively, and dried in an oven. The dried solid was suspended in hydrochloric acid (37.4%, 0.5 mL) and methanol (40 mL) and refluxed for 16h to remove CTAB, thus obtaining MSN-NH 2. After the reflux, MSN-NH 2 was washed with ethanol and MSN-NH 2 was dried in an oven. MSN-NH 2 was characterized by TEM, BET nitrogen adsorption and desorption, and Zeta potential.
Construction of clustered fluorescent amplifying module
In order to ligate DNA onto the surface of MSN-NH 2, this example first carboxylates MSN-NH 2. Specifically, MSN-NH 2 (50 mg) is ultrasonically dispersed in N, N-dimethylformamide (DMF, 15 mL), succinic anhydride (500 mg) is added, and after stirring reaction is carried out for 8 hours, the mesoporous silicon nanoparticle (MSN-COOH) with carboxylated surface can be obtained. MSN-COOH was separated from the reaction system by centrifugation (12000 rpm,6 min) and washed 3 times with water, and then resuspended in N-morpholinoethanesulfonic acid (MES) buffer (MES 100mM,pH 6.0,5.0mL).
The specific steps of Rh6G loading are as follows: rh6G (5.0 mg) was dissolved in water (10.0 mL) with stirring to give a Rh6G solution. Next, MSN-COOH (2 mL,10.0mg mL -1) suspension was added to the Rh6G solution described above, and stirred in the dark for 24h to give a Rh 6G-loaded MSN-COOH red solid (Rh6G@MSN-COOH). After centrifugation of the red solid, rapid washing with a small amount of water, rh6g@msn-COOH was collected again by centrifugation.
The connecting chain is modified to the surface of Rh6G@MSN-COOH through an amide condensation reaction. Specifically, rh6G@MSN-COOH suspension (1.0 mg) was taken, EDC (3.0 mg) and sulfoNHS (8.0 mg) were added, and the reaction was stirred for 20min to activate carboxyl groups. Next, PBS buffer (1.0 mL) and an aminated linker chain (100. Mu.M, 60. Mu.L) were added and reacted in the dark for 24 hours to obtain a clustered fluorescent amplification module (Rh6G@MSN-LINKING STRAND). The obtained Rh6G@MSN-LINKING STRAND is washed twice by TE buffer solution and stored for standby.
Feasibility verification of aptamer sensor detection of ATP
To verify whether the aptamer sensor can be used for detection of cancer markers, the present example uses ATP as a model and an overall experiment was performed. First, a PBSM suspension of MBD-recognition probe (0.5 mg mL -1, 10. Mu.L) was taken, and after shaking, ATP (50. Mu.M) was added and incubated at room temperature for 40min. Then, under the action of an externally applied magnetic field, the MBD-recognition probe after the ATP incubation is separated from the reaction system (15 min). MBD-recognition probe was washed with TE buffer, dispersed in ligation buffer (Tris 20mM, naCl 60 mM), added with Rh6G@MSN-LINKING STRAND, and mixed on a four-dimensional mixer at room temperature for 25min. Then separating the MBD and Rh6G@MSN-LINKING STRAND captured by the MBD under the action of an externally applied magnetic field. After washing the separated solid with TE buffer, TE buffer was added and the temperature was raised to 80℃for 25min. After centrifugation, the supernatant was aspirated and the fluorescence emission spectrum of the supernatant was collected. And simultaneously collecting fluorescence emission spectra of the aptamer sensor without ATP under the same experimental condition. And recording a fluorescence emission spectrum with the emission wavelength in the range of 545-595 nm under the excitation wavelength of 525nm, and taking a fluorescence signal value at the 550nm maximum emission wavelength of Rh6G as the fluorescence signal intensity of the aptamer sensor. The fluorescence signal intensities in this chapter were all measured using this parameter.
Experimental condition optimization
In order to demonstrate optimal detection performance when the aptamer sensor is used for ATP detection, the present example examines several key experimental conditions in aptamer sensor detection, including Mg 2+ concentration, ATP binding time, DNA ligation time, release temperature, na + concentration, and rh6g@msn-LINKING STRAND concentration. Specifically, this example collected fluorescence emission spectra of different Mg 2+ concentrations (0, 2.0, 4.0, 6.0, 8.0, and 10 mM), ATP binding times (10, 20, 30, 40, 50, 60 min), release temperatures (40, 50, 60, 70, 80, 90 ℃), DNA ligation times (5, 10, 15, 20, 25, 30 min), na + concentrations (15, 30, 45, 60, 75, and 90 mM), and rh6g@msn-LINKING STRAND concentrations (10, 20, 30, 40, 50, 60 μg mL -1) aptamer sensors incubated with ATP. The fluorescence signal intensity of the aptamer sensor in the presence (positive group) and absence (negative group) of ATP was measured. And selecting the experimental condition with the largest ratio of the two as the optimal experimental condition for detecting the ATP by the aptamer sensor.
Specificity investigation of aptamer sensor detection ATP
TTP, CTP, GTP and dATP are selected as interferents, and the specificity of ATP detection by the aptamer sensor is examined. Specifically, target ATP in the feasibility verification of detecting ATP by the aptamer sensor was replaced with TTP, CTP, GTP and dATP with 5-fold concentrations, and after the reaction was performed according to the experimental protocol, the fluorescence signal intensity of the aptamer sensor was measured. The specificity of the aptamer sensor for detecting ATP is examined by comparing the difference in fluorescence signal intensity of the aptamer sensor.
Versatility investigation of aptamer sensors
Thrombin (as a protein target model) and PDGF-BB (as a dual aptamer target model) were selected to examine the versatility of the aptamer sensor. The experimental procedure for thrombin detection is identical to that for ATP. The experimental procedure for PDGF-BB assay is as follows: PDGF-BB was added to a mixture of MBD-aptamer probe 1 and Rh6G@MSN-aptamer probe 2 in PBSM buffer, and mixed on a four-dimensional mixer at room temperature for 40min. Under the action of an externally applied magnetic field, the MBD and Rh6G@MSN-aptamer probe 2 captured by the MBD are separated from a reaction system (15 min), washed by a TE buffer solution, added with the TE buffer solution and heated to 80 ℃ for 25min. After centrifugation, the supernatant was collected and the fluorescence emission spectrum of the supernatant was collected.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.
Example 1 construction and characterization of aptamer sensors
As shown in FIG. 2A, TEM images show that the synthesized MSN-NH 2 is a porous spherical particle with an average particle size of about 80.3nm, as measured. The BET nitrogen adsorption and desorption isotherms are typical type iv curves (FIG. 2B), indicating that MSN-NH 2 has a mesoporous structure, whose average pore diameter is calculated to be 2.3nm (FIG. 2C). TEM image of MSN-NH 2 and BET nitrogen adsorption and desorption result show that spherical nano particles with mesoporous structure are successfully synthesized.
The present example describes the transmission electron microscope characterization of MBD-SA. TEM images showed (FIG. 2D) that MBD-SA is spherical particles with an average diameter of about 1.1 μm. After incubating MBD-recognition probe with ATP and MSN-LINKING STRAND, TEM images showed that a large amount of MSN appeared on the surface of the beads (fig. 2E), indicating that mbd@msn-LINKING STRAND was successfully prepared, indicating that MSN-LINKING STRAND could be captured by MBD-recognition probe incubated with ATP, consistent with design principles. To evidence this, the surface Zeta potentials of MSN-NH 2, MSN-COOH, MSN-LINKING STRAND, MBD-SA and MBD@MSN-linking probe (FIG. 2F) were further determined to be +10.6eV, -33.2eV, -24.9eV, -10.4eV and-25.5 eV, respectively. Because the amino group is easy to be protonated to generate positive charges, the Zeta potential of the surface of the MSN-NH 2 is positive; after carboxylation and DNA modification, the surface Zeta potentials of MSN-COOH and MSN-LINKING STRAND become negative values, which are consistent with the inherent electronegativity of carboxyl and DNA, indicating that MSN is successfully carboxylated and DNA modified; then, MBD-SA has negative value due to the electronegative surface Zeta potential of the surface streptavidin; after the MBD-recognition probe is incubated with ATP and MSN-LINKING STRAND, the Zeta potential of the surface becomes relatively positive and is close to that of MSN-LINKING STRAND, indicating that the MB surface is successfully connected with MSN-LINKING STRAND. The above results demonstrate successful fabrication of aptamer sensors.
Example 2 an aptamer sensor for ATP detection
First, the feasibility of interactions between nucleic acid strands for ATP detection was verified by polyacrylamide gel electrophoresis (PAGE). As shown in FIG. 3A, the bands in lanes 1-5 represent the aptamer, anchor DNA, recognition probe, ligation strand, anchor DNA-ligation strand hybrid, respectively. After mixing the recognition probe and the ligation strand, lane 6 shows the same band as the recognition probe (lane 3) and the ligation strand (lane 4), indicating that no hybridization reaction between the recognition probe and the ligation strand occurs when ATP is not present. Lane 7 shows the electrophoresis result of the mixed recognition probe, ligation strand and ATP, and shows the same band as lane 1 and lane 5, indicating that the hybridization of the anchored DNA and ligation strand is triggered by the binding of ATP and aptamer after the three are mixed. The above results are in accordance with the design principle, indicating that the interactions between the nucleic acid strands meet the design requirements.
The present example then verifies the feasibility of the aptamer sensor for ATP detection using fluorescence. Fluorescence emission spectra of the aptamer sensor in the presence (positive condition) and absence (negative condition) of ATP were collected, respectively, as shown in fig. 3B. The fluorescence emission profile of the aptamer sensor is significantly higher under positive conditions than under negative conditions. Under negative conditions, the fluorescence signal intensity of the aptamer sensor at the maximum fluorescence emission peak (550 nm) of Rh6G is 334.0; under positive conditions, the aptamer sensor has significantly enhanced fluorescence signal intensity here (2812). The fluorescence signal intensity under positive conditions was 8.4 times that under negative conditions, indicating that the aptamer sensor was able to generate a significantly enhanced fluorescence signal in response to ATP, which can be used to detect ATP.
Experimental condition optimization
In order to obtain optimal ATP detection performance, the present embodiment optimizes several key experimental conditions of the aptamer sensor. As shown in FIG. 4, the present example measures the fluorescence signal intensities of the aptamer sensor in the presence (positive group) and absence (negative group) of ATP, respectively, and the two bar graphs in the histogram represent the fluorescence signal intensities of the positive group (denoted by F) and the negative group (denoted by F 0) under different experimental conditions, respectively. The broken line is a ratio curve of F/F 0 under different experimental conditions. In the embodiment, the experimental condition with the largest F/F 0 ratio is selected as the optimal experimental condition of the aptamer sensor for ATP detection.
Mg 2+ concentration affects the binding efficiency of ATP and aptamer, so this example first examined the performance impact of Mg 2+ concentration in the reaction buffer on the detection of ATP by the aptamer sensor. As shown in fig. 4A, with increasing Mg 2+ concentration, both F and F 0 values increase, since increasing Mg 2+ concentration favors aptamer folding into a stable binding conformation, competing binding reactions of ATP with aptamer occur more readily, favoring exposure of anchored DNA and capture of the clustered fluorescent amplification module; however, increasing Mg 2+ concentrations at the same time may result in folding of the non-specific aptamer into a binding conformation, resulting in an increase in fluorescence signal intensity under negative conditions. Under this influence, the F/F 0 ratio increased and then decreased with increasing Mg 2+ concentration, with the maximum occurring at a Mg 2+ concentration of 6.0mM. Thus, the optimal Mg 2+ concentration of the reaction buffer when the aptamer sensor detects ATP was determined to be 6.0mM.
The binding time of ATP and aptamer affects the progress of competing binding reaction, and thus the capture efficiency of the clustered fluorescent amplification module. Therefore, this example next examined the effect of the binding time of ATP on the detection performance of the aptamer sensor when detecting ATP. As shown in fig. 4B, the F and F 0 values increase with increasing ATP binding time, indicating that extending binding time helps to increase capture efficiency, but also results in some non-specific capture. The effect is that the F/F 0 ratio increases first and then decreases slowly, the maximum occurring at 40min. Thus, the optimal ATP binding time for the aptamer sensor for detecting ATP was determined to be 40min.
The heating temperature is a key factor affecting Rh6G release, and the Rh6G release efficiency affects the sensitivity of the aptamer sensor when detecting ATP, so the performance impact of the Rh6G release temperature on the aptamer sensor when detecting ATP was examined. The values of F and F 0 increase with increasing release temperature, while the ratio of F/F 0 shows a trend of increasing before stabilizing (FIG. 4C). This is because the temperature rise is advantageous for the release of Rh6G in the clustered fluorescent amplification module, and the release rate of Rh6G does not rise any more when a certain temperature is reached. The F/F 0 ratio tended to stabilize after 80℃and therefore the optimum release temperature was chosen to be 80 ℃.
The time of attachment of the anchor DNA to the ligation strand affects the efficiency of MBD capture rh6g@msn-LINKING STRAND, and thus the performance of the aptamer sensor to detect ATP, thus in turn optimizing the time of attachment of the two. As shown in FIG. 4D, the values of F and F 0 increase with increasing DNA ligation time, since the prolonged time is advantageous for improving the ligation efficiency of the anchor DNA to the ligation strand, but at the same time may also result in a certain non-specific ligation. As a result, the maximum value of the F/F 0 ratio occurs at 25min, and thus the optimal DNA ligation time was determined to be 25min.
The concentration of Na + during ligation affects the stability of double strand during ligation, and this example examined the effect of Na + concentration of ligation buffer on the detection performance of the aptamer sensor when used to detect ATP during DNA ligation. As shown in FIG. 4E, the maximum value of F/F 0 occurs at a Na + concentration of 60mM and the optimal Na + concentration of the ligation buffer is determined to be 60mM.
Finally, this example examined the effect of Rh6G@MSN-LINKING STRAND concentration on the detection performance of an aptamer sensor for detecting ATP. As shown in FIG. 4F, the values of F and F 0 increase with increasing Rh6G@MSN-LINKING STRAND concentration, because increasing the concentration of the clustered fluorescent amplification modules promotes the capture efficiency of the magnetic separation probe module, but at the same time may cause some non-specific capture. The maximum value of F/F 0 occurs at 40 μg mL -1, and the final determination of 40 μg mL -1 is the optimal Rh6G@MSN-LINKING STRAND concentration for use of the aptamer sensor for ATP detection.
Based on the above results, the optimal experimental conditions for the aptamer sensor were determined to be an aptamer binding buffer Mg 2+ concentration of 6.0 mM, an ATP binding time of 40min, an Rh6G release temperature of 80 ℃, a ligation time of 25min for the ligation strand to the anchor DNA, a ligation buffer Na + concentration of 60mM, and a rh6g@msn-LINKING STRAND concentration of 40 μg mL -1. The tests in the following examples were all performed under optimal conditions.
Sensitivity of aptamer sensor for ATP detection
To examine the sensitivity of the aptamer sensor for ATP detection, this example measured the fluorescence emission spectra of aptamer sensors for different concentrations of ATP sample treatment. As shown in FIG. 5A, the fluorescence signal intensity of the aptamer sensor gradually increases as the concentration of ATP (0-50. Mu.M) increases after the reaction according to the experimental protocol, because the cluster amplification module captured by the aptamer sensor increases as the concentration of ATP increases. The net fluorescence signal intensity at 550nm (difference between the fluorescence signal intensity under positive conditions and the fluorescence signal intensity under negative conditions, F-F 0) versus ATP concentration curve is shown in FIG. 5B, which shows a tendency to rise first and then gently. This is probably because after the ATP concentration reaches a certain value, the ATP aptamer is bound by ATP entirely, and further increases in ATP concentration do not expose more of the anchored DNA to capture the clustered fluorescent amplification module to generate a fluorescent signal. FIG. 5B (inset) shows a working curve of F-F 0 and ATP concentration showing that F-F 0 and ATP concentration are well-linear in the range of 10nM to 1.0. Mu.M, with the linear equation being F-F 0＝1603CATP+7.4(R2 =0.998. The LOD of the aptamer sensor to detect ATP was calculated to be 2.1nM according to the limit of detection (LOD) calculation formula lod=3σ/K, where σ is the standard deviation of three parallel measurements for a blank sample and K is the calibration slope of a linear fit curve. This example compares this limit of detection with some reported ATP detection strategies, which is lower than the limit of detection for most reported ATP detection strategies (table 4), indicating that the aptamer sensor has good sensitivity for ATP detection. This benefits from the design of clustered fluorescent amplification, which allows for the conversion of an ATP binding event into a cluster (dye molecules in a clustered fluorescent amplification module) of fluorescent signals, thus exhibiting a stronger signal amplification capability and higher sensitivity.
Table 4 compares with reported ATP detection methods
/>
Specificity of aptamer sensor for ATP detection
In this example TTP, CTP, GTP and dATP were selected as interferents and the specificity of the aptamer sensor for ATP detection was examined. TTP, CTP, GTP is a nucleotide, dATP is a deoxynucleotide, and there is only a difference in base or ribotype from ATP, which is typical in the investigation of specificity of ATP detection. As shown in fig. 6, the fluorescence signal of the aptamer sensor after co-incubation with ATP was significantly enhanced compared to the blank sample with PBS buffer alone as a control; under the same experimental conditions, after the aptamer sensor is incubated with TTP, CTP, GTP, dATP times of concentration, the fluorescence signal intensity is not significantly different from that of a blank sample. The results indicate that the aptamer sensor only produces significantly enhanced fluorescence signal intensity for ATP response, with good specificity when used for ATP detection. The reason that aptamer sensors exhibit good specificity is that the high selectivity of aptamer binding to target, interferents cannot bind to the aptamer, and subsequent amplification cannot be triggered.
Precision and reproducibility of aptamer sensor for ATP detection
To examine the precision and reproducibility of the aptamer sensor for ATP detection, three concentrations (20, 100, and 500 nM) of ATP samples were selected for the experiment in this example. And respectively measuring the fluorescent signal intensity of the aptamer sensor after the three samples are reacted in parallel according to an experimental scheme, and then calculating the daily precision and the daytime precision of the aptamer sensor. Firstly, the accuracy of the aptamer sensor in detecting ATP is examined. Specifically, the fluorescence signal intensities of the three concentrations of ATP incubated with the aptamer sensor were measured, each set of samples was measured three times in parallel, the corresponding ATP concentrations were converted according to the linear equation in fig. 5B (inset), and the relative standard deviations of ATP concentrations (RSD, n=3) were calculated, 5.1%, 2.8% and 2.1%, respectively, indicating that the method has good daily precision. Then, the daytime accuracy of the aptamer sensor for detecting ATP was examined in a similar manner. The fluorescence signal intensities of the three groups of samples are respectively measured within three days, and the RSD of the concentration measurement values are respectively 5.5%, 4.9% and 2.9% after conversion into ATP concentration, so that the method has good daytime precision and reproducibility.
Performance of aptamer sensor in detecting ATP in human serum system
To verify the feasibility of the sensor in biological samples, the embodiment uses human serum to simulate actual biological samples, and examines the performance of the aptamer sensor in detecting ATP in complex systems. The influence of complex components in serum on the aptamer sensor is examined by comparing the difference of fluorescence signal intensities of ATP detected by the aptamer sensor in a human serum system and a reaction buffer. First, this example detects three concentrations (20, 100, and 500 nM) of ATP samples in reaction buffer and 10-fold diluted human serum system, respectively, and compares the difference in fluorescence signal intensity of the aptamer sensor. As shown in FIG. 7, the aptamer sensor has no obvious difference between the fluorescence signal intensity in the human serum system and the fluorescence signal intensity in the reaction buffer solution, which indicates that the aptamer sensor has detection performance close to that of the buffer solution system when being used for detecting ATP in the human serum system, and indicates that the aptamer sensor has higher resistance to interference of complex components in the human serum system and has certain application potential in detection of actual biological samples. In addition, in this example, the aptamer sensor was tested for ATP recovery in human serum. The three concentrations of ATP are respectively added into a human serum system, then the fluorescence signal intensity of the aptamer sensor is measured after the reaction is carried out according to an experimental scheme, and the recovery rate is calculated according to a standard curve. As shown in Table 5, the labeled recovery rates of the three concentrations of ATP samples are 97%, 101% and 101%, and the RSD is 4.7%, 4.4% and 1.5%, respectively, which indicate that the aptamer sensor has good anti-interference capability and has certain application potential in the analysis of actual biological samples.
TABLE 5 labelling recovery for ATP detection in human serum system
Example 2 an aptamer sensor for detection of cancer markers
Finally, this example examined the versatility of the aptamer sensor for cancer marker detection using thrombin (as a protein target model) and PDGF-BB (as a dual aptamer target model) as models (see fig. 8 and 9, respectively, for the principle).
This example demonstrates the feasibility of an aptamer sensor for thrombin and PDGF-BB detection. First, the feasibility of interactions between designed nucleic acid strands was verified by PAGE. As shown in FIG. 8B, the PAGE pattern shows the interactions between the nucleic acid strands used for thrombin detection. The bands in lanes 1,2, 3, 4,5 represent the aptamer, anchor DNA, recognition probe, ligation strand, and hybridization of the anchor DNA to the ligation strand, respectively. Lane 6 shows the result of electrophoresis after mixing the recognition probe and the ligation strand, and shows that the same band as the positions of the recognition probe (lane 3) and the ligation strand (lane 4) is present, indicating that no hybridization reaction occurs between the recognition probe and the ligation strand in the absence of thrombin. Lane 7 shows the result of electrophoresis after mixing the recognition probe, the ligation strand and thrombin, and shows the same band as in lane 5, which is a band hybridized between the anchor DNA and the ligation strand; in addition, a band with a slower migration rate was present, which is a band generated by binding of the aptamer to thrombin, indicating that thrombin can bind to the aptamer. The electrophoresis result shows that thrombin can generate competitive binding reaction with the aptamer in the recognition probe and trigger the hybridization of the connecting strand and the anchored DNA, which is consistent with the design principle. The designed interactions between the nucleic acid chains and the thrombin and between the nucleic acid chains meet the design requirements, and can be used for detecting the thrombin.
Next, this example also demonstrates the feasibility of interactions between nucleic acid strands for PDGF-BB detection using the PAGE method. As shown in FIG. 9B, the bands in lanes 1 and 2 represent aptamer probe 1 and aptamer probe 2, respectively. Lanes 3 and 4 are the electrophoresis results of the aptamer probe 1 and the aptamer probe 2 mixed with PDGF-BB, respectively. Lanes 3 and 4 show a slow migration band, indicating that both aptamer probes bind PDGF-BB. Lane 5 shows the result of electrophoresis after mixing of the aptamer probe 1 and the aptamer probe 2, in which two bands appear at the same positions as the aptamer probe 1 (lane 1) and the aptamer probe 2 (lane 2), respectively, indicating that no hybridization reaction between the aptamer probe 1 and the aptamer probe 2 occurs in the absence of PDGF-BB. Lane 6 shows the electrophoresis results after mixing of aptamer probe 1, aptamer probe 2 and PDGF-BB. The bands of aptamer probe 1 and aptamer probe 2 disappeared in this lane because the aptamer probe reacted with PDGF-BB; a slow migration rate band was also present, which was generated by combining aptamer probe 1, aptamer probe 2 and PDGF-BB. The gel electrophoresis experiment result shows that PDGF-BB can be combined with two aptamer probes, the interaction between the probes and the PDGF-BB meets the design requirement, and the PDGF-BB can be used for PDGF-BB detection.
The overall feasibility of aptamer sensors for thrombin and PDGF-BB detection was verified using fluorescence. There is a significant difference in the fluorescence emission spectra of the aptamer sensor in the presence and absence of thrombin. Under the negative condition, the fluorescence signal intensity of the aptamer sensor at 550nm of the Rh6G maximum emission peak is 301.5; after thrombin addition, the fluorescence signal intensity was significantly increased (2732) at 9.1 times the fluorescence signal intensity under negative conditions. The results show that the aptamer sensor can respond to thrombin to show obvious fluorescence signal enhancement, and can be used for detecting thrombin. Similarly, the fluorescence signal intensity of the aptamer sensor after PDGF-BB addition was 5.6 times higher than that without PDGF-BB addition, indicating that the aptamer sensor was suitable for PDGF-BB detection. The aptamer sensor can be used for detecting various target objects only by replacing the identification unit, and the aptamer sensor has universality when used for detection. This is because the modular design enhances the independence between the functional modules of the aptamer sensor, thereby increasing the flexibility of detection.
The sensitivity of the aptamer sensor to detect thrombin and PDGF-BB was then examined in this example. As shown in FIG. 10A, the fluorescence signal intensity of the aptamer sensor gradually increased as the thrombin concentration increased in the range of 0-20nM after the reaction according to the experimental protocol. The fluorescence signal intensity at 550nm was used to plot the F-F 0 versus thrombin concentration, which showed a trend of rising followed by flattening (FIG. 10B). This is because after the thrombin concentration reaches a specific value, the thrombin aptamer is fully involved in binding, and further increases in thrombin concentration do not expose more anchored DNA and capture the clustered fluorescent amplification module, resulting in higher fluorescent signal intensities. FIG. 10B (inset) is a working curve of F-F 0 and thrombin concentration showing that F-F 0 and thrombin concentration exhibit good linearity over the 20pM-4.0nM range, with a linear equation of F-F 0＝509.1 CThrombin-8.0(R2 =0.996. According to the equation of lod=3σ/K, the present embodiment calculates an aptamer sensor
Table 6 compares with reported thrombin detection methods
The LOD for thrombin detection was 4.1pM. This example compares this limit of detection with some reported thrombin detection strategies, which is lower than the limit of detection for most reported thrombin detection methods (Table 6). The results show that the aptamer sensor has good sensitivity when used for thrombin detection, which is also beneficial to the clustered fluorescent amplification design, and the design converts a small amount of target binding events into a large amount of clustered signals, so that the sensitivity of the aptamer sensor when used for detection is improved.
As shown in FIG. 11A, in the range of 0-20nM, the intensity of the fluorescent signal of the aptamer sensor also tended to increase gradually as PDGF-BB concentration increased and the reaction was performed according to the experimental protocol. The fluorescence signal intensity at 550nm was used to plot the concentration of F-F 0 and PDGF-BB (FIG. 11B), which was elevated and then gradually gentle. The reason is that after the PDGF-BB concentration reaches a certain value, all PDGF-BB aptamer probes participate in binding, and the subsequent amplification cannot be triggered by continuously increasing the PDGF-BB concentration, so that higher fluorescence signal intensity is generated. FIG. 11B (inset) shows a working curve of F-F 0 and PDGF-BB concentrations, with F-F 0 and PDGF-BB concentrations exhibiting good linearity over the 10pM-2.0nM range, with a linear equation of F-F 0＝320.4CPDGF-BB+4.8(R2 =0.995). According to the LOD calculation formula, the embodiment calculates that the LOD of the aptamer sensor for detecting PDGF-BB is 2.4pM. This limit of detection is lower than the limit of detection of most of the reported PDGF-BB detection strategies (Table 7), indicating good sensitivity of the aptamer sensor when detecting with PDGF-BB. The high sensitivity of the aptamer sensor for detecting thrombin and PDGF-BB is also beneficial to the design of clustered fluorescent amplification, so that a small amount of target binding events are converted into a large amount of signals, and the signal amplification capability of the aptamer sensor is improved.
Table 7 compares PDGF-BB detection methods reported
Finally, in order to examine the performance of the aptamer sensor in detecting thrombin and PDGF-BB in a complex system, the present example performed a labeled recovery test on the aptamer sensor in detecting thrombin and PDGF-BB in a human serum system, respectively. First, this example adds three concentrations (0.04, 0.40 and 4.0 nM) of thrombin samples to a 10-fold diluted human serum system, then after performing a reaction according to an experimental protocol, measures the fluorescence signal intensity, and calculates recovery and RSD values according to a standard curve. As shown in Table 8, the labeled recovery rates of the three thrombin samples are 96%, 95% and 98%, and the RSD is 6.6%, 4.6% and 3.9%, respectively, which indicate that the aptamer sensor can resist the interference of complex matrixes in a human serum system when being used for thrombin detection, and has certain application potential in thrombin analysis of the complex system. Next, the present example measured the standard recovery of three concentrations (0.02, 0.2 and 2.0 nM) of PDGF-BB samples. After the reaction according to the experimental protocol, the fluorescence signal intensity of PDGF-BB was measured and the recovery and RSD values were calculated graphically. The labeled recovery rates of PDGF-BB were 95%, 103% and 102%, and the RSD was 5.2%, 2.8% and 2.8%, respectively (Table 9), indicating that the aptamer sensor has better anti-interference capability when used for PDGF-BB detection in human serum samples. The experimental results show that the aptamer sensor has a certain practical biological sample analysis potential.
Table 8 labeled recovery of thrombin detection in human serum system
TABLE 9 labelling recovery for PDGF-BB detection in human serum System
In summary, the aptamer sensor can be used for detecting thrombin and PDGF-BB by replacing the corresponding recognition probes by plug and play; the aptamer sensor has higher sensitivity when used for detecting thrombin and PDGF-BB, and has better recovery rate when detecting the target in a human serum system. Experimental results show that the aptamer sensor has universality when used for detecting cancer markers.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> University of Shandong
<120> A general aptamer biosensor and application thereof in the field of marker detection
<130> 2010
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 33
<212> DNA
<213> Artificial sequence
<400> 1
gagagaacct gggggagtat tgcggaggaa ggt 33
<210> 2
<211> 25
<212> DNA
<213> Artificial sequence
<400> 2
cccaggttct ctctcacaca tctgt 25
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence
<400> 3
gagagaacct ggggatatgc tagactatcg 30
<210> 4
<211> 15
<212> DNA
<213> Artificial sequence
<400> 4
cgatagtcta gcata 15
<210> 5
<211> 35
<212> DNA
<213> Artificial sequence
<400> 5
gctcggagtc cgtggtaggg caggttgggg tgact 35
<210> 6
<211> 25
<212> DNA
<213> Artificial sequence
<400> 6
acggactccg agctcacaca tctgt 25
<210> 7
<211> 30
<212> DNA
<213> Artificial sequence
<400> 7
gctcggagtc cgtgatatgc tagactatcg 30
<210> 8
<211> 15
<212> DNA
<213> Artificial sequence
<400> 8
cgatagtcta gcata 15
<210> 9
<211> 47
<212> DNA
<213> Artificial sequence
<400> 9
caggctacgg cacgtagagc atcaccatga tcctgtcaca catctgt 47
<210> 10
<211> 52
<212> DNA
<213> Artificial sequence
<400> 10
caggctacgg cacgtagagc atcaccatga tcctggatct actagactat cg 52
<210> 11
<211> 12
<212> DNA
<213> Artificial sequence
<400> 11
cgatagtcta gc 12
Claims (6)
1. A universal aptamer biosensor, wherein the biosensor comprises a magnetic probe module and a signal amplification module; the magnetic probe module is a magnetic bead for modifying an identification probe, and the identification probe is provided with an aptamer sequence of a detection target;
the signal amplification module comprises a porous carrier, wherein the porous carrier is used for encapsulating fluorescent dye, and the surface of the porous carrier is modified with a connecting chain, and the connecting chain comprises an antisense sequence of an identification probe or an aptamer sequence;
the magnetic bead surface is modified with streptavidin, and the recognition probe is connected to the magnetic bead surface by means of the specific combination of the streptavidin and biotin;
the recognition probe comprises an anchor DNA and an aptamer:
The 3' end of the aptamer is provided with an aptamer sequence for combining with a detection target; the proper length of the aptamer sequence is 20-40 bases; the 5 'end of the anchoring DNA is provided with a complementary sequence of a connecting chain, the 3' end of the anchoring DNA is modified with biotin, and the anchoring DNA is connected to the surface of the magnetic bead through the specific combination of the biotin and streptavidin; the 5 'end of the connecting strand is provided with a complementary sequence of anchored DNA, the 3' end is provided with an amino group which is connected to the surface of the carrier with holes through an amide bond, and the other sites are blocked through a blocking strand;
the universal aptamer biosensor is used for ATP detection, and the specific sequence is as follows:
An aptamer: GAGAGAACCTGGGGGAGTATTGCGGAGGAAGGT;
anchoring the DNA: CCCAGGTTCTCTCTCACACATCTGT-biotin;
Connecting chain: GAGAGAACCTGGGGATATGCTAGACTATCG-NH 2;
Closed chain: CGATAGTCTAGCATA;
or, the universal aptamer biosensor is a universal aptamer biosensor for thrombin detection, and the specific sequence is as follows;
An aptamer: GCTCGGAGTCCGTGGTAGGGCAGGTTGGGGTGACT;
Anchoring the DNA: ACGGACTCCGAGCTCACACATCTGT-biotin;
connecting chain: GCTCGGAGTCCGTGATATGCTAGACTATCG-NH 2;
Closed chain: CGATAGTCTAGCATA.
2. The universal aptamer biosensor as claimed in claim 1, wherein said signal amplification module:
The porous carrier is a spherical material with hollow inside and through holes distributed on the surface of the carrier, the diameter is 1-2 mu m, the aperture is 1-5 nm, and the surface can be fixedly connected with a chain in a physical and chemical mode;
the spherical carrier is an aminated mesoporous silicon nanoparticle and is connected with the connecting chain through an amide bond;
Further, the mesoporous silicon nanoparticle adopts a copolycondensation method, and the preparation method comprises the following steps: slowly adding CTAB aqueous solution into alkali liquor, stirring and heating for reaction for a period of time, dropwise adding TEOS and a silane coupling agent into the reaction solution, and continuously heating for reaction to obtain a white solid; separating the white solid, washing and drying, and then adding the white solid into hydrochloric acid and methanol solution for refluxing to remove CTAB, thus obtaining MSN-NH 2;
the porous carrier internally encapsulates fluorescent dye, wherein the fluorescent dye comprises, but is not limited to, rhodamine Rh6G, rhodamine B, triphenylamine and one of derivatives thereof.
3. The universal aptamer biosensor of claim 2, wherein the mesoporous silicon nanoparticle surface modification of the connecting chains is achieved by amide bond connection; specifically, the connection mode is as follows: carboxylating the MSN-NH 2, and modifying a connecting chain to the surface of mesoporous silicon particles through amide condensation;
specifically, the preparation method of the signal amplification module comprises the following steps: adding succinic anhydride into a dimethylformamide solution of MSN-NH 2 to react to obtain the mesoporous silicon nanoparticle with carboxylated surface; continuously adding the Rh6G solution, stirring in a dark place to obtain an MSN-COOH red solid carrying Rh6G, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and thiosuccinimide into the suspension, stirring for reaction to activate carboxyl, adding PBS buffer solution and an aminated connecting chain, and reacting in a dark place to obtain the signal amplification module.
4. The universal aptamer biosensor of claim 1, wherein,
The proper length of the aptamer sequence is 25-40 bases.
5. Use of the universal aptamer biosensor of any one of claims 1-4 in the field of marker detection;
The application is as follows:
(1) For the preparation of diagnostic products.
6. The use of a universal aptamer biosensor as claimed in claim 5 in the field of detection of markers,
In the application of the aspect (1), the diagnostic product includes, but is not limited to, diagnostic kits, diagnostic chips and other possible diagnostic systems.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210198403.6A CN114561446B (en) | 2022-03-01 | 2022-03-01 | Universal aptamer biosensor and application thereof in field of marker detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210198403.6A CN114561446B (en) | 2022-03-01 | 2022-03-01 | Universal aptamer biosensor and application thereof in field of marker detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114561446A CN114561446A (en) | 2022-05-31 |
| CN114561446B true CN114561446B (en) | 2024-04-26 |
Family
ID=81714985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210198403.6A Active CN114561446B (en) | 2022-03-01 | 2022-03-01 | Universal aptamer biosensor and application thereof in field of marker detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114561446B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101752048A (en) * | 2008-12-17 | 2010-06-23 | 中国科学院大连化学物理研究所 | Ordered short-channel magnetic mesoporous material |
| CN103743805A (en) * | 2014-01-17 | 2014-04-23 | 湖南大学 | Biosensor based on nitrogen-hybridized mesoporous carbon as well as preparation method and application of biosensor |
| CN104178568A (en) * | 2014-07-25 | 2014-12-03 | 清华大学 | Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe |
| CN108007991A (en) * | 2017-11-30 | 2018-05-08 | 中南大学 | A kind of biology sensor and its construction method for Electrochemical Detection PDGF-BB |
| CN110257482A (en) * | 2019-07-16 | 2019-09-20 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of blood coagulation enzyme assay method based on aptamer and telomere enzymatic amplification |
| CN113311153A (en) * | 2021-05-12 | 2021-08-27 | 华中科技大学 | Multifunctional nanoparticle for integrated diagnosis and treatment of Alzheimer disease |
| CN113687074A (en) * | 2021-08-26 | 2021-11-23 | 四川大学华西医院 | Thrombin detection method based on magnetic separation deoxyribozyme and cyclic cleavage and thrombin kit |
-
2022
- 2022-03-01 CN CN202210198403.6A patent/CN114561446B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101752048A (en) * | 2008-12-17 | 2010-06-23 | 中国科学院大连化学物理研究所 | Ordered short-channel magnetic mesoporous material |
| CN103743805A (en) * | 2014-01-17 | 2014-04-23 | 湖南大学 | Biosensor based on nitrogen-hybridized mesoporous carbon as well as preparation method and application of biosensor |
| CN104178568A (en) * | 2014-07-25 | 2014-12-03 | 清华大学 | Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe |
| CN108007991A (en) * | 2017-11-30 | 2018-05-08 | 中南大学 | A kind of biology sensor and its construction method for Electrochemical Detection PDGF-BB |
| CN110257482A (en) * | 2019-07-16 | 2019-09-20 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of blood coagulation enzyme assay method based on aptamer and telomere enzymatic amplification |
| CN113311153A (en) * | 2021-05-12 | 2021-08-27 | 华中科技大学 | Multifunctional nanoparticle for integrated diagnosis and treatment of Alzheimer disease |
| CN113687074A (en) * | 2021-08-26 | 2021-11-23 | 四川大学华西医院 | Thrombin detection method based on magnetic separation deoxyribozyme and cyclic cleavage and thrombin kit |
Non-Patent Citations (2)
| Title |
|---|
| Target-Induced Displacement Reaction Accompanying Cargo Release from Magnetic Mesoporous Silica Nanocontainers for Fluorescence Immunoassay;Dianping Tang等;Dianping Tang等;第85卷(第21期);摘要、图1 * |
| 基于纳米多孔材料的电化学DNA传感器的研究与应用;赵聪林;中国优秀硕士学位论文全文数据库 基础科学辑(第12期);第44页第2-326 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114561446A (en) | 2022-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yoo et al. | Detection and beyond: Challenges and advances in aptamer-based biosensors | |
| Kong et al. | Label-free fluorescence turn-on aptasensor for prostate-specific antigen sensing based on aggregation-induced emission–silica nanospheres | |
| US20170159113A1 (en) | Methods and compositions for detection of analytes | |
| CN101424642B (en) | Target molecule detecting method based on nanometer aurum and nucleic acid structure | |
| JP2006528183A (en) | Assay for protease enzyme activity | |
| CN109762558A (en) | A kind of preparation method for the Ratiometric fluorescent probe of PPi content in quantitative detection urine | |
| JP2012242394A (en) | Kits for detecting target material and methods of detecting target material using the kits | |
| Li et al. | Multi-functional crosslinked Au nanoaggregates for the amplified optical DNA detection | |
| Zhong et al. | Expanding the scope of chemiluminescence in bioanalysis with functional nanomaterials | |
| CN112725343A (en) | Protein marker detection kit combining gold nanoprobe and CRISPR-Cas and detection method | |
| Zhang et al. | Recent development and applications of sensors for the detection of matrix metalloproteinases | |
| CN111235229A (en) | Ratiometric fluorescent probe for detecting PTK7 and preparation method thereof | |
| Wang et al. | Simultaneous ultrasensitive ADP and ATP quantification based on CRISPR/Cas12a integrated ZIF-90@ Ag3AuS2@ Fe3O4 nanocomposites | |
| CN107674675A (en) | Detection targets preparation method of carbon quantum dot turn on probes and products thereof and application with folacin receptor | |
| Geng et al. | Research progress on preparation methods and sensing applications of molecularly imprinted polymer-aptamer dual recognition elements | |
| Wang et al. | DNA coated CoZn-ZIF metal-organic frameworks for fluorescent sensing guanosine triphosphate and discrimination of nucleoside triphosphates | |
| CN114561446B (en) | Universal aptamer biosensor and application thereof in field of marker detection | |
| CN112979530A (en) | DNA-adsorbable nanoparticle, nucleic acid detection probe, and preparation methods and applications thereof | |
| CN110132946B (en) | Aptamer sensor and preparation method and application thereof | |
| Yang et al. | Ultrasensitive fluorescence detection of sequence-specific DNA via labeling hairpin DNA probes for fluorescein o-acrylate polymers | |
| CN108760695B (en) | Method for quantitatively detecting thrombin by using phosphorescence probe based on PRET | |
| Park et al. | Highly photoluminescent superparamagnetic silica composites for on-site biosensors | |
| CN113607947B (en) | Method for detecting alpha fetoprotein by using aptamer based on aggregation-induced emission mark and azide functionalized single-walled carbon nanotube | |
| Hu et al. | Target-triggering, signal-amplified chemo/bio-sensors based on aggregation-induced emission luminogens | |
| Zheng et al. | Study on an electrochemical biosensor for thrombin recognition based on aptamers and nano particles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |