CN106749423A - 一种顺铂荧光配合物及其应用 - Google Patents
一种顺铂荧光配合物及其应用 Download PDFInfo
- Publication number
- CN106749423A CN106749423A CN201611054092.7A CN201611054092A CN106749423A CN 106749423 A CN106749423 A CN 106749423A CN 201611054092 A CN201611054092 A CN 201611054092A CN 106749423 A CN106749423 A CN 106749423A
- Authority
- CN
- China
- Prior art keywords
- platinum
- cis
- fluorescence
- mark
- protein
- 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.)
- Pending
Links
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 67
- 238000002372 labelling Methods 0.000 claims abstract description 53
- 239000002773 nucleotide Substances 0.000 claims abstract description 29
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 29
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 21
- 201000010099 disease Diseases 0.000 claims abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 238000003745 diagnosis Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 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 16
- 239000003550 marker Substances 0.000 claims description 13
- 229960002685 biotin Drugs 0.000 claims description 8
- 235000020958 biotin Nutrition 0.000 claims description 8
- 239000011616 biotin Substances 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000013399 early diagnosis Methods 0.000 claims description 2
- 238000004393 prognosis Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 35
- 108090000623 proteins and genes Proteins 0.000 abstract description 33
- 239000000523 sample Substances 0.000 abstract description 16
- 238000001514 detection method Methods 0.000 abstract description 8
- 150000007523 nucleic acids Chemical class 0.000 abstract description 6
- 230000000052 comparative effect Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 238000009396 hybridization Methods 0.000 abstract description 4
- 238000002493 microarray Methods 0.000 abstract description 4
- 108020004707 nucleic acids Proteins 0.000 abstract description 4
- 102000039446 nucleic acids Human genes 0.000 abstract description 4
- 108091005461 Nucleic proteins Proteins 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 238000010189 synthetic method Methods 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 28
- 238000000034 method Methods 0.000 description 24
- 108020004414 DNA Proteins 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 15
- 150000001875 compounds Chemical class 0.000 description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 12
- 229920002477 rna polymer Polymers 0.000 description 10
- 239000002253 acid Substances 0.000 description 9
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 9
- 238000007901 in situ hybridization Methods 0.000 description 9
- 230000001640 apoptogenic effect Effects 0.000 description 8
- 210000001766 X chromosome Anatomy 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 6
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 210000002593 Y chromosome Anatomy 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 206010005003 Bladder cancer Diseases 0.000 description 5
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 5
- 229960005156 digoxin Drugs 0.000 description 5
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- 208000010570 urinary bladder carcinoma Diseases 0.000 description 5
- 108020003215 DNA Probes Proteins 0.000 description 4
- 239000003298 DNA probe Substances 0.000 description 4
- 229920002301 cellulose acetate Polymers 0.000 description 4
- 210000002230 centromere Anatomy 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002777 nucleoside Substances 0.000 description 4
- 125000003835 nucleoside group Chemical group 0.000 description 4
- 238000005191 phase separation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 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 description 4
- 239000000243 solution Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- HOKKHZGPKSLGJE-GSVOUGTGSA-N N-Methyl-D-aspartic acid Chemical compound CN[C@@H](C(O)=O)CC(O)=O HOKKHZGPKSLGJE-GSVOUGTGSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 229920002873 Polyethylenimine Polymers 0.000 description 3
- 241000238370 Sepia Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 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 3
- 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 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 2
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 2
- 208000005156 Dehydration Diseases 0.000 description 2
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102100022630 Glutamate receptor ionotropic, NMDA 2B Human genes 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 108010038912 Retinoid X Receptors Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000218636 Thuja Species 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 201000001531 bladder carcinoma Diseases 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 125000001309 chloro group Chemical group Cl* 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229950004590 digitalin Drugs 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical class NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- RVZPDKXEHIRFPM-UHFFFAOYSA-N tert-butyl n-(6-aminohexyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCCCCCN RVZPDKXEHIRFPM-UHFFFAOYSA-N 0.000 description 2
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 2
- 239000001226 triphosphate Substances 0.000 description 2
- 235000011178 triphosphate Nutrition 0.000 description 2
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 229940005561 1,4-benzoquinone Drugs 0.000 description 1
- UZGKAASZIMOAMU-UHFFFAOYSA-N 124177-85-1 Chemical compound NP(=O)=O UZGKAASZIMOAMU-UHFFFAOYSA-N 0.000 description 1
- 238000004294 195Pt NMR spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- BHNQPLPANNDEGL-UHFFFAOYSA-N 2-(4-octylphenoxy)ethanol Chemical compound CCCCCCCCC1=CC=C(OCCO)C=C1 BHNQPLPANNDEGL-UHFFFAOYSA-N 0.000 description 1
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 description 1
- HVGAOLHAWVNIQU-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;pyrrolidine-2,5-dione Chemical compound O=C1CCC(=O)N1.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 HVGAOLHAWVNIQU-UFLZEWODSA-N 0.000 description 1
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000018899 Glutamate Receptors Human genes 0.000 description 1
- 108010027915 Glutamate Receptors Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical class OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- IYKNGWWPNRDDHK-ONEGZZNKSA-N [[5-[2,4-dioxo-5-[(e)-3-[5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl)pentanoylamino]prop-1-enyl]pyrimidin-1-yl]-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)CC1N1C(=O)NC(=O)C(\C=C\CNC(=O)CCCCC2C3NC(=O)NC3CS2)=C1 IYKNGWWPNRDDHK-ONEGZZNKSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- XLJMAIOERFSOGZ-UHFFFAOYSA-N anhydrous cyanic acid Natural products OC#N XLJMAIOERFSOGZ-UHFFFAOYSA-N 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 230000023077 detection of light stimulus Effects 0.000 description 1
- CKNOLMVLQUPVMU-YMMLYESFSA-N digitalin Chemical group C1([C@@H]2[C@@]3(C)CC[C@H]4[C@H]([C@]3(C[C@@H]2O)O)CC[C@H]2[C@]4(C)CC[C@@H](C2)O[C@H]2[C@H](O)[C@H]([C@H]([C@@H](C)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)OC)=CC(=O)OC1 CKNOLMVLQUPVMU-YMMLYESFSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- MJJFTDMZSMPNCL-UHFFFAOYSA-M lithium;2-hydroxy-3,4-diiodobenzoate Chemical compound [Li+].OC1=C(I)C(I)=CC=C1C([O-])=O MJJFTDMZSMPNCL-UHFFFAOYSA-M 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000001216 nucleic acid method Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 150000003058 platinum compounds Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- HHFOOWPWAXNJNY-UHFFFAOYSA-N promoxolane Chemical compound CC(C)C1(C(C)C)OCC(CO)O1 HHFOOWPWAXNJNY-UHFFFAOYSA-N 0.000 description 1
- 229950008352 promoxolane Drugs 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- XREMTGLZTIREIM-UHFFFAOYSA-N tert-butyl 2,7-diaminoheptanoate Chemical compound CC(C)(C)OC(=O)C(N)CCCCCN XREMTGLZTIREIM-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- -1 tetramethyl isocyanic acid Chemical compound 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0086—Platinum compounds
- C07F15/0093—Platinum compounds without a metal-carbon linkage
-
- 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
- C12Q1/6841—In situ hybridisation
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明提供了一种顺铂荧光配合物,还涉及其合成方法以及其在制备核苷酸和蛋白质探针中的应用。该顺铂荧光配合物既能与荧光素相连接,又能与DNA、RNA、蛋白质牢固连接,从而能够作为生物探针的连接主体,十分适用于目标DNA、RNA和蛋白质的标记与检测;该顺铂荧光配合物标记操作步骤简单,不受标记分子大小的限制,不受标记混合体系的pH值的影响;可以广泛用于核苷酸、蛋白质(抗体)、核酸和蛋白质微阵列(Micro Arrays),比较基因组杂交等细分领域;此外,其显著提高了标记效率,缩短了标记时间并增加了荧光强度稳定性,取得了优异的标记效果;因此,其在核苷酸和蛋白质探针的制备、生物医药研发、疾病的诊断等领域具有良好的应用前景。
Description
技术领域
本发明属于生物化学领域,涉及一种顺铂荧光配合物,还涉及该顺铂荧光配合物在制备核苷酸和蛋白质探针中的应用。
背景技术
荧光素是具有共轭双键体系化学结构的化合物,当受到特定波长光照射时,被激发成为激发态,从激发态恢复到基态过程中可发出荧光。荧光素的这一特性,被广泛运用于生物学和医学领域的检测中。荧光素在生物学和医学领域中的运用始于上世纪80年代末,目前,在生物学和医学领域,荧光素主要用于标记核苷酸和蛋白质。具体分述如下:
核苷酸(DNA,RNA)的荧光素标记技术,主要包括:(1)酶促生物素标记核酸法:主要是以生物素化的脱氧核苷三磷酸(Bio-11-dUTP,Bio–7–dATP、Bio-11-dCTP等)经DNA聚合酶作用掺入DNA。Bio-dUTP代替dTTP,Bio-dATP代替dATP,Bio-dCTP代替dCTP。(2)末端标记法:主要分为5’-磷酸的化学标记法和3’-OH的酶促标记法。5’-磷酸的化学标记法是在寡核苷酸上的5’-磷酸上接一个乙二胺,然后用琥珀酰亚胺生物素,将生物素基团连接在磷酸酰胺基上。3’-OH的酶促标记法是用末端转移法将Bio-11–dUTP加于其3’-OH端(脱去一个焦磷酸)。(3)酶标DNA法:使用辣根过氧化酶(HRP)或碱性磷酸酶(AP)作为标记试剂。通过对苯醌(PBQ)与聚乙烯亚胺(PEI)连接成(HRP-PBQ-PEI+),此试剂在戊二醛的作用下与变性的DNA结合,使HRP与DNA连接在一起,组成HRP标记的DNA探针。(4)酶标寡核苷酸法:包括核苷酸5’末端标记HRP法和内部标记AP法。核苷酸5’末端标记HRP法是在HRP中产生一个-HS反应基团,在寡核苷酸合成终了加在5’端,带一个C6的-HS基,与活化的HRP反应生成5’-HRP寡核苷酸。内部标记AP法是在全成寡核苷酸过程中将一个5’带连接臂及CF3基团的尿苷3’亚磷酰亚胺合成在寡核苷酸链中,合成后此活化的寡核苷酸与AP反应即得到AP标记的寡核苷酸。(5)DNA半抗原标记法:其原理与Bio-11-dTUP相同,只是用毛地黄甙代替生物素形成Dig–11-dUTP,酶促掺入DNA分子。用抗毛地黄甙抗体检测标记在DNA上的半抗原分子(地高辛)。
此外,常用的核苷酸链荧光标记技术主要包括缺口平移法和随机引物标记法两种。
其中,所述缺口平移法是指:在DNA合成酶的作用下,随机在双链DNA分子的一条链上随机切开若干个缺口,在3’—OH端逐个加入新的核苷酸,同时切除5’端游离的核苷酸,导致了新链合成缺口的平移,新链核苷酸的合成是以另一互补链为模板,按碱基互补的原则合成;在新链合成中用上述方法标记dNTP(dATP,dGTP,dCTP,dUTP),标记荧光的核苷酸就被掺入到新链中,达到核苷酸链标记目的。
其中,所述随机引物标记法是指:在Klenow酶催化下,通过随机引物结合目标核苷酸序列,以模板链为参照合成互补链的方法。在此过程中,用荧光素、生物素、半抗原(地高辛)等标记过的脱氧核苷三磷酸dNTP(dATP,dGTP,dCTP,dUTP)掺入到核苷酸新链中,从而使靶核苷酸链(DNA链)得以高效标记。
此外,蛋白质的标记主要是通过荧光素衍生化反应来实现的。其主要原理是,蛋白质结构中的一些功能性氨基酸基团与荧光素以共价键相连接,从而使目的蛋白质被标记。通常在以蛋白质氨基端作为衍生化位点,其上的氢原子在碱性条件下被荧光基团取代,且反应条件相对温和,因此,是目前常用的蛋白质标记方法。
目前,以上标记方法已被广泛应用于核酸和蛋白质标记的研究和医学诊断中,有些已作为临床诊断和评估的指标。例如:应用荧光原位杂交探针,原位杂交探针,抗体和蛋白质标记技术检测各种疾病中相关指标的改变。目前常用的荧光素一般为FITC,罗丹明等。但是,现有技术中的标记方法仍然存在标记效率不高、标记时间长和荧光强度不稳定等缺陷。
因此,如何设计并合成出一种新的配合物,使得其既能与荧光素相连接,又能与脱氧核糖核酸(DNA)、核糖核酸(RNA)、蛋白质牢固连接,以期用该配合物作为生物探针的连接主体,应用于目标DNA、RNA和蛋白质的检测。
发明内容
针对上述现有技术中存在的缺陷与不足,发明人合成了一种新的以金属铂为骨架的配位化合物,并发明了以之为介导的核苷酸或蛋白质的标记新方法;该方法实现以金属铂为骨架的配位化合物为介导,牢固连接荧光素和靶分子(核苷酸或蛋白质),从而达到荧光标记的目的。
具体地,本发明第一方面提供了一种顺铂荧光配合物,具有如下结构式:
其中,n=3,4,5,6,7,8,9;
其中,Marker为标记基团,至少包括荧光素。
优选地,上述顺铂荧光配合物中,所述标记基团还包括:生物素和/或半抗原。其中,半抗原例如为地高辛。
值得说明的是,本发明所提供的顺铂荧光配合物中,金属Pt原子分别与3个氮原子和1个氯原子配位,当其与核苷酸或蛋白质相结合时,氯原子会被脱去而与核苷酸或蛋白质上的结合位点形成共价键。具体地,所述顺铂荧光配合物可以连接生物素,并连接地高辛等半抗原化合物,也可以直接连接荧光素(如罗丹明,FITC,DEAC,Cy5,Cy3),同时,可牢固地连接鸟嘌呤(Guanine)氮原子N7位点,产生共价键牢固结合,达到标记核酸链(DNA链,RNA链)的目的。基于上述特点,本发明所述的顺铂荧光配合物能用于对荧光原位杂交(Fluorescence in situ hybridization,FISH),原位杂交(in situ Hybridization,ISH),比较基因组杂交(comparative genomic hybridization,CGH)以及基因芯片中的核苷酸链进行荧光或者染料标记。而在所述顺铂荧光配合物与蛋白质结合时,具体的结合位点为:例如,所述顺铂荧光配合物与甲硫氨酸(Methionine),半胱氨酸(Cysteine)侧链上的硫原子形成共价键,和组氨酸(Histidine)分子上的氮原子形成稳定的化学结构。
优选地,上述顺铂荧光配合物中,所述标记基团选自以下A~I中的任一种:
本发明的第二方面,还提供了一种本发明第一方面所述顺铂荧光配合物在制备核苷酸和蛋白质探针中的应用。
本发明的第三方面,还提供了一种本发明第一方面所述顺铂荧光配合物在制备试剂盒中的应用,其中,所述试剂盒用于疾病的早期诊断、治疗随诊、预后或复发诊断。
本发明所提供的顺铂荧光配合物,既能与荧光素相连接,又能与脱氧核糖核酸(DNA)、核糖核酸(RNA)、蛋白质牢固连接,从而能够作为生物探针的连接主体,十分适用于目标DNA、RNA和蛋白质的检测。相较于现有技术中的标记方法,本发明所提供的顺铂荧光配合物用于核苷酸和蛋白质标记具有如下优点:(1)标记速度快:经多次实验表明,完成对DNA标记的平均时间不超过45分钟,完成对RNA标记的平均时间不超过20分钟,完成对蛋白质(抗体)标记的平均时间不超过30分钟;(2)操作步骤简单,避免使用传统的酶标记技术,操作条件不苛刻,使用方便;(3)对石蜡标本,福尔马林包埋组织的标记效果显著;(4)不受标记分子大小的限制,理论上对于Micro RNA(约20bp)至大片段核酸探针(>20kb)都能进行有效标记;不受标记混合体系的pH值的影响;可以广泛用于核苷酸、蛋白质(抗体)、核酸和蛋白质微阵列(Micro Arrays),比较基因组杂交等细分领域。
综上所述,所述顺铂荧光配合物在用于标记核苷酸或蛋白质时,显著提高了标记效率,缩短了标记时间并增加了荧光强度稳定性,取得了优异的标记效果;因此,本发明所提供的顺铂荧光配合物在核苷酸和蛋白质探针的制备、生物医药研发、疾病的诊断等领域具有良好的应用前景。
附图说明
图1显示了本发明所述的顺铂荧光配合物的结构式,并示出了其标记基团与用于连接核算或蛋白质的部位;
图2为n=6时本发明所述的顺铂荧光配合物的合成路线图;
图3为n=3,标记基团分别为E,C,G,I时,所述顺铂荧光配合物的浓度(X轴)与荧光强度(Y轴)的关系曲线图;
图4为n=6,标记基团分别为E,C,G,I时,所述顺铂荧光配合物的浓度(X轴)与荧光强度(Y轴)的关系曲线图;
图5为n=9,标记基团分别为E,C,G,I时,所述顺铂荧光配合物的浓度(X轴)与荧光强度(Y轴)的关系曲线图;
图6为所述顺铂荧光配合物结合生物素(n=6)、亲和素以及Cy5标记的荧光抗体进行标记后,所形成的生物素-亲和素-荧光抗体复合物的浓度(X轴)与荧光强度(Y轴)的关系曲线图;
图7为实施例4中所实施的铂-荧光素标记方法用于标记的荧光原位杂交探针在精子染色体中的荧光检测图;其中(1000倍油镜下),蓝色代表DAPI复染的精子核部分,红色代表精子Y染色体,绿色信号代表X染色体(原图为彩色,此处为灰度图);
图8为实施例5中所标记的精子的荧光检测图,其中(1000倍油镜下),绿色代表荧光染料FITC发出的荧光,荧光信号明亮,该荧光集中在精子尾部,精子头部较少染色(原图为彩色,此处为灰度图);
图9为实施例6对照组中的顺铂荧光配合物标记抗体标记膀胱癌T24细胞凋亡的显微图,其中,棕褐色表示凋亡细胞,蓝色表示正常细胞,因此可见凋亡细胞极少(原图为彩色,此处为灰度图);
图10为实施例6实验组中的顺铂荧光配合物标记抗体标记膀胱癌T24细胞凋亡的显微图,其中,棕褐色表示凋亡细胞,蓝色表示正常细胞,因此可见大量的凋亡细胞(原图为彩色,此处为灰度图)。
具体实施方式
下面结合具体实施方式对本发明作进一步阐述,但本发明并不限于以下实施方式。
实施例1
n=6时本发明所述的顺铂荧光配合物的合成
如图2所示的合成路线,具体步骤如下:
(1)以1,6-二氨基己烷作为起始反应物;首先将(Boc)2O(12.3g,0.056m ol)溶解在100mL二氯甲烷中,然后缓慢滴加溶有1,6-二氨基己烷(33g,0.28mol)的150mL二氯甲烷溶液,0℃下反应4小时,然后室温下反应20小时;反应完全后,用4×100mL饱和食盐水洗涤反应液,分液,有机相用无水硫酸钠干燥,减压蒸去溶剂,得到14.3g无色油状液体,即N-Boc-1,6-二氨基己烷(N-Boc-1,6-diaminohexane,中间体a),无需纯化,直接进行下一步反应。
(2)将[Pt(en)Cl2](500mg,1.53mmol)溶解在50ml DMF中,然后加入AgNO3(248mg,1.46mmol,0.95equiv),避光室温搅拌16小时,过滤除去AgCl,将化合物a(315mg,<0.8equiv)溶解在25mL正己烷中,加入到上述滤液中,40℃下反应过夜,减压除去溶剂,柱色谱分离纯化,得到白色粉末,即中间体b([Pt(II)(N-tert-Butoxycarbonyl-1,6-hexanediamine)(en)Cl](NO3))(650mg收率74%),1H NMR(D2O):δ=3.11(2H),2.71(4H),2.63(2H),1.69(2H),1.52(2H),1.46(9H),1.39(4H)ppm;195Pt-NMR:δ=-2625ppm。
(3)将化合物b(28mg,0.05mmol)溶解在2mL三氟乙酸中,50℃下过夜反应,然后用5M NaOH溶液调节到中性,过滤,干燥得到中间体c,将中间体c溶解在一定量的DMF中,加入1.2equiv EDC和三乙胺,室温下反应12小时,反应结束后柱色谱分离得到最终产物。
实施例2
本实验将铂与罗丹明,DEAC,gloxalic acid,FITC,TRITC,Alexa fluor 594等荧光素进行标记,并对荧光强度进行测定。结果提示顺铂为介导的配合物能与以上荧光素很好地结合,发射荧光。(本实验使用的顺铂荧光配合物的n=3,6,9)。将marker-Pt配位化合物溶解在0.1M三羟甲基氨基甲烷的缓冲溶液中,pH=7.1,室温下对荧光强度进行测定。有机顺铂荧光化合物(碳链长度分别是n=3,n=6,n=9)的荧光强度和配合物浓度之间的关系如图3~5所示。
我们还采用有机顺顺铂荧光配合物结合生物素,并且使用亲和素、标记Cy5的荧光抗体进行标记,形成生物素-亲和素-荧光抗体复合物,荧光信号得到显著放大。其结果见图6。
实施例3
采用本发明所述的顺铂荧光配合物制备核酸和蛋白质探针的实验
一、核苷酸标记方法
将100ug的纯化的靶核苷酸克隆与2ug纯化后的铂-生物素-荧光素(FITC,Cy5)标记物(本实验采用顺铂荧光配合物中的n=6)混合,80℃反应约40min(反应时间根据靶分子类型可做适当调整,RNA反应时间可以缩短至20min左右),吸出标记样本的混合物,经过上述醋酸纤维素液相分离柱,得到纯化的荧光素化的核苷酸序列。
二、蛋白质分子探针标记方法
将100ug的蛋白质(本实验所用为抗体IgG)与1ug纯化后的铂-生物素-荧光素(FITC,Cy5)标记物(本实验采用顺铂荧光配合物中的n=6)混合,37℃反应约20min后,吸出标记样本的混合物,经过液相分离柱,得到纯化的荧光素化的抗体(蛋白质)。
结构式如下:
其中,复合物中的Marker可以被异硫氰酸荧光素(fluorescein isothiocyanate,FITC),罗丹明(Rohdamine),四甲基异氰酸罗达明(tetrametrylrhodarnineisothiocyante,TRITC),Cy5,Cy3标记,该复合物也可以与半抗原类物质(如地高辛Digoxin)以及其他细胞染料等化合。
最终产物的分离和纯化:使用醋酸纤维素液相色谱分离柱,在均相条件下合成了醋酸纤维素,将其涂渍在修饰硅胶表面,制备涂渍型高效液相色谱固定相。将合成后的产物装入色谱柱,控制压力为40Mpa左右,高效液相色谱正相色谱条件下,对产物进行高效分离。
三、采用本发明所述的顺铂荧光配合物制备的标记探针实验结果
(1)评价标记手段:通过检测探针的荧光信号值(F),确定含有荧光基团的单核苷酸量(s),并以此计算出该种核苷酸的总量(n),最终可获得探针DNA的量(w)。以总的探针DNA的质量(w)、或以总的探针DNA的质量(w)除以模板mRNA或DNA的质量(r)的比值e来表示荧光标记效率,可表示为:w=4×324.5×n=1298×(mk+1)×s;e=(w/r)×100%=(1298×(mk+1)×s/r)×100%。(2)标记效率结果:将已被克隆纯化的人类乳腺癌Her-2基因,运用本发明标记技术(有机顺铂标记)标记法进行标记效率进行实验比较,实验组顺铂荧光配合物按照碳链长度分为n=3,n=6,n=9三组,每组共重复进行5次标记实验。实验结果用SPSS14.0软件进行统计分析。当碳链长度n=3时,顺铂荧光配合物标记效率达到(2.563±0.461)%;当碳链长度n=6时,顺铂荧光配合物标记效率达到(2.865±0.229)%;当碳链长度n=9时,顺铂荧光配合物标记效率达到(2.665±0.327)%,结果表明:顺铂荧光配合物(n=3,6,9)三组之间标记效率无显著差异(p>0.05)。
(3)标记时间结果:运用顺铂荧光配合物标记法对人类乳腺癌Her-2克隆基因进行标记,其中实验组顺铂荧光配合物按照碳链长度分为n=3,n=6,n=9三组。每组共重复进行5次标记实验。统计标记所需时间差异,结果用SPSS14.0软件进行分析。当碳链长度n=3时,顺铂荧光配合物标记方法所用的平均标记时间为(40±3.42)分钟;当碳链长度n=6时,顺铂荧光配合物标记方法所用的平均标记时间为(32.2±5.69)分钟;当碳链长度n=9时,顺铂荧光配合物标记方法所用的平均标记时间为(42.8±6.21)分钟。顺铂荧光配合物标记方法三组之间(碳链n=3,6,9时),三者差异有显著性(p<0.05),当碳链长度n=6时,标记时间更短。
(4)标记靶分子长度比较结果:采取X染色体着丝粒寡核苷酸重复序列和P53基因克隆进行铂-荧光素方法标记(采用碳链长度n=3,n=6,n=9三组进行实验)。X染色体探针序列长度42bp(5’GACGATGGAGTTAACTCAGGGAGCT GAACATTCGTTATGATG3’),P53基因长度约为20kb,按照(n=3,n=6,n=9)分3组对X着丝粒序列和P53基因分别标记,每组重复实验10次,并用FI TC标记的铂复合物进行标记反应,反应时间40min,温度82℃。反应完成后将混合物经过醋酸纤维素液相色谱柱,40Mpa高压分离过柱纯化。将纯化后的标记产物在紫外分光光度计检测下,控制检测波长480nm左右,统计吸光度,并按照上述评价标记手段进行标记长度结果统计。实验结果使用SPSS14.0软件分析。本发明方法对小片段的X染色体DNA探针标记率:当n=3时,标记效率达到(2.660±0.501)%;n=6时,标记效率达到(2.792±0.483)%;n=9时,标记效率达到(2.611±0.495)%。标记人类P53基因(>20kb)的标记效率:当n=3时,标记效率达到(2.632±0.596)%;当n=6时,标记效率达到(2.807±0.310)%,当n=9时,标记效率达到(2.718±0.441)%。n=3,6,9时,X着丝粒片段组相对P53组其标记效率均无统计学差异(p>0.05)。由此可见,该标记技术能高效标记核酸片段,并且不受片段长度影响。
实施例4
采用本发明所述顺铂荧光配合物介导标记的寡核苷酸荧光原位杂交探针检测人类精子染色体的试验(荧光原位杂交技术(FISH))
针对X,Y染色体着丝粒区域的高度串联重复片段(Satellite III,Alpha Satellite),设计DNA寡核苷酸探针序列,具体序列如下:
X染色体着丝粒序列
5’GACGATGGAGTTAACTCAGGGAGCTGAACATTCGTTATGATG3’
5’ATTTCTTTGGAATCGGGAGTCAATATTTCCACAG3’
Y染色体着丝粒序列
5’TCCATTGGAGTCAATTCTACGCTTTCGACACCCA3’
5’CCAGTCGAATCCATTCTGCAGAGTACATACC3’
5’CTGCATACAATTTCATGCCTCCATTCGTTCCCA3’
用合适的合成方法合成上述DNA序列(X,Y染色体),取合成产物(约2OD值),加100ul去离子水后与纯化的荧光素标记物混合,其中含Y染色体D NA序列用铂-罗丹明(红色)标记物1ug;含X染色体DNA序列用铂-FITC(绿色)标记物1ug(本实验采用所述顺铂荧光配合物中碳链长度n=6)。将混合物80℃反应40min,取出,进液相分离柱纯化待用。
将精子细胞常规固定,经过二硫苏糖醇(DTT),二碘水杨酸锂(LIS)处理后,于75℃下变性5min,取出经70%-80%-90%乙醇脱水,将标记的DNA探针经92℃变性10min后,20℃处理30min,取出,滴加于精子玻片杂交区域,封片,37℃杂交过夜。取出玻片,经过0.4×SSC/0.3%Nonidet-P40溶液清洗,梯度脱水,晾干,DAPI复染,荧光显微镜(Olympus BX51)观察。
结果显示:荧光信号明显,特异性高,无弥散信号,其中红色代表精子Y染色体着丝粒区域,绿色代表X染色体着丝粒区域,以铂为介导的复合物标记方法能够有效对DNA进行标记,且被标记的荧光探针同样具有较满意的荧光强度(见图7)。
实施例5
采用本发明所述的顺铂荧光配合物为介导的荧光素标记抗体,检测人类精子一种谷氨酸受体,N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)进行抗原抗体特异反应的试验(蛋白质抗体荧光标记)
自Sigma公司采购抗NMDA抗体,以及二抗。将100ug的二抗IgG与1u g纯化后的铂-生物素-FITC标记物混合(本实验采用所述顺铂荧光配合物中的n=6),37℃反应约20min后,吸出标记样本的混合物,40Mpa经过液相分离柱,得到纯化的荧光素化的抗体备用。
人类精子样品从21-43岁男子节欲3天后手淫获得。精子于37℃液化30分钟,检测到正常精子,在含4%多聚甲醛的磷酸盐缓冲液中固定30分钟,涂于明胶包被的载玻片上,干燥、固定。人类精子涂片用0.01M PBS洗涤,然后用含10%山羊或兔血清及0.1%TritonX-100的PBS缓冲液封闭以降低非特异性免疫染色。用初级抗体或作为阴性对照的正常非免疫血清4℃过夜,孵化后,涂片用PBS漂洗3次,并与已经标记好的相应的二抗(铂-FITC)结合,室温下孵化2小时,然后用PBS漂洗,最后涂片在荧光显微镜下检测并拍照。
结果显示:实验发现在精子尾部可清楚地观察到NR2B特异性的绿色荧光(FITC)但精子头部无免疫活性(见图8),说明NR2B受体主要存在于精子的尾部,从而证明铂介导的荧光标记技术同样能有效对蛋白质抗体进行标记。
实施例6
采用本发明所述顺铂荧光配合物为介导的荧光素标记抗体标记膀胱癌T24细胞凋亡的试验
取膀胱癌T24细胞,加入木黄酮40mg/L。分别于12、24、36、48h收获膀胱癌T24细胞,用TUNEL法检测凋亡细胞,观察木黄酮对T24细胞的影响,加药48h后收获细胞,离心、弃上清液。具体按试剂盒说明书进行操作,加入0.01mol/L PBS混匀,将细胞悬液滴于经二甲蓝处理的载玻片上,放置20min,载玻片放入4%多聚甲醛中过夜,PBS及双蒸水洗片3次,加标记缓冲液,室温下放置15min,加混合液及标记缓冲液;37℃于湿盒中标记60min,其中标记物为本发明的新化合物与地高辛-Pt-dUTP复合物(本实验采用所述顺铂荧光配合物中的n=6),常规封片,于光学显微镜下观察细胞并拍照,细胞染成棕褐色为凋亡细胞,蓝色为正常细胞。图9显示对照组活细胞呈蓝色,凋亡细胞极少;图10显示,实验组见大量染成棕色的凋亡细胞,该铂-复合物介导的标记手法同样可以用于组织化学染色,且能很好显色。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (5)
1.一种顺铂荧光配合物,其特征在于,具有如下结构式:
其中,n=3,4,5,6,7,8,9;
其中,Marker为标记基团,至少包括荧光素。
2.根据权利要求1所述的顺铂荧光配合物,其特征在于,所述标记基团还包括:生物素和/或半抗原。
3.根据权利要求1或2所述的顺铂荧光配合物,其特征在于,所述标记基团选自以下A~I中的任一种:
4.一种根据权利要求1~3中任一项所述的顺铂荧光配合物在制备核苷酸和蛋白质探针中的应用。
5.一种根据权利要求1~3中任一项所述的顺铂荧光配合物在制备试剂盒中的应用,其特征在于,所述试剂盒用于疾病的早期诊断、治疗随诊、预后或复发诊断。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611054092.7A CN106749423A (zh) | 2016-11-25 | 2016-11-25 | 一种顺铂荧光配合物及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611054092.7A CN106749423A (zh) | 2016-11-25 | 2016-11-25 | 一种顺铂荧光配合物及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN106749423A true CN106749423A (zh) | 2017-05-31 |
Family
ID=58911458
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201611054092.7A Pending CN106749423A (zh) | 2016-11-25 | 2016-11-25 | 一种顺铂荧光配合物及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106749423A (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110711250A (zh) * | 2019-09-25 | 2020-01-21 | 南京市口腔医院 | 一种双靶向多模协同治疗纳米载药体系的构建方法 |
CN111875611A (zh) * | 2020-05-21 | 2020-11-03 | 湖南师范大学 | 一种抗癌铂前药还原活化的荧光探针及其制备方法与应用 |
CN111978308A (zh) * | 2020-08-06 | 2020-11-24 | 宁波海关技术中心 | 一种试剂盒的制备方法及其在蛋白质荧光标记上的应用 |
CN112094302A (zh) * | 2020-11-05 | 2020-12-18 | 江苏申基生物科技有限公司 | 一种细胞内顺铂检测探针及其制备方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002097439A2 (en) * | 2001-05-28 | 2002-12-05 | Kreatech Biotechnology B.V. | A differential labelling method |
-
2016
- 2016-11-25 CN CN201611054092.7A patent/CN106749423A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002097439A2 (en) * | 2001-05-28 | 2002-12-05 | Kreatech Biotechnology B.V. | A differential labelling method |
Non-Patent Citations (2)
Title |
---|
DAVID HERNANDEZ-SANTOS ET AL.: ""Genosensor Based on a Platinum(II) Complex as Electrocatalytic Label"", 《ANAL. CHEM.》 * |
R. J. HEETEBRIJ ET AL.,: ""Platinum(II)-Based Coordination Compounds as Nucleic Acid Labeling Reagents: Synthesis, Reactivity, and Applications in Hybridization Assays"", 《CHEMBIOCHEM》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110711250A (zh) * | 2019-09-25 | 2020-01-21 | 南京市口腔医院 | 一种双靶向多模协同治疗纳米载药体系的构建方法 |
CN110711250B (zh) * | 2019-09-25 | 2021-10-26 | 南京市口腔医院 | 一种双靶向多模协同治疗纳米载药体系的构建方法 |
CN111875611A (zh) * | 2020-05-21 | 2020-11-03 | 湖南师范大学 | 一种抗癌铂前药还原活化的荧光探针及其制备方法与应用 |
CN111978308A (zh) * | 2020-08-06 | 2020-11-24 | 宁波海关技术中心 | 一种试剂盒的制备方法及其在蛋白质荧光标记上的应用 |
CN111978308B (zh) * | 2020-08-06 | 2023-08-18 | 宁波海关技术中心 | 一种试剂盒的制备方法及其在蛋白质荧光标记上的应用 |
CN112094302A (zh) * | 2020-11-05 | 2020-12-18 | 江苏申基生物科技有限公司 | 一种细胞内顺铂检测探针及其制备方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4361138B2 (ja) | 蛍光シアニン色素 | |
US7157572B2 (en) | UV excitable energy transfer reagents | |
US4898951A (en) | Compounds used as intermediates in the preparations of non-radioactive biological probes | |
US10647740B2 (en) | Multisignal labeling reagents and processes and uses therefor | |
EP0292128A1 (en) | Improved DNA probes | |
CN101605743A (zh) | 用于产生报告分子的点击化学 | |
CN106749423A (zh) | 一种顺铂荧光配合物及其应用 | |
JPH09500525A (ja) | 核酸配列を増幅し、検出するための化学的方法 | |
MX2007013296A (es) | Nuevas estrategias de etiquetado para la deteccion sensible de analitos. | |
CA2341520C (en) | Energy transfer dyes | |
JPH05508862A (ja) | 大きな櫛型分枝状ポリヌクレオチド | |
CN1902490B (zh) | 生物分子检测方法及其所用的标记染料和标记试剂盒 | |
CN106459001A (zh) | 多次甲基化合物和其作为荧光标记物的用途 | |
JP3008690B2 (ja) | リガンドまたはレセプターの測定法 | |
EP1546391A2 (en) | Energy transfer dyes, terminators and use thereof | |
ES2352509T3 (es) | Nuevas estrategias de marcaje para la detección sensible de analitos. | |
US6967250B1 (en) | Energy transfer dyes | |
Satusky et al. | Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA | |
JP3679691B2 (ja) | 新規なピリリウム化合物、その製造方法、それを含む核酸染色剤、および標識核酸 | |
JP2002281978A (ja) | Dnaプローブアレイを用いた標的dnaのノンラベル検出法 | |
JP2002107364A (ja) | ノンラベル検出用生物試薬アレイ及びその作製方法 | |
JP2001247857A (ja) | 蛍光性物質および蛍光標識方法 | |
JPH05502577A (ja) | Hbvのための改良した増幅核酸ハイブリダイゼーションアッセイ |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |
|
RJ01 | Rejection of invention patent application after publication |