CN116462639B - 一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用 - Google Patents
一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用 Download PDFInfo
- Publication number
- CN116462639B CN116462639B CN202310415928.5A CN202310415928A CN116462639B CN 116462639 B CN116462639 B CN 116462639B CN 202310415928 A CN202310415928 A CN 202310415928A CN 116462639 B CN116462639 B CN 116462639B
- Authority
- CN
- China
- Prior art keywords
- apn
- lan
- probe
- fluorescent
- fluorescence
- 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
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 41
- 108010049990 CD13 Antigens Proteins 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 11
- 238000004737 colorimetric analysis Methods 0.000 claims abstract description 10
- 238000002795 fluorescence method Methods 0.000 claims abstract description 5
- 238000003745 diagnosis Methods 0.000 claims description 10
- 230000000007 visual effect Effects 0.000 claims description 5
- 239000000523 sample Substances 0.000 abstract description 126
- 238000001514 detection method Methods 0.000 abstract description 30
- 238000003384 imaging method Methods 0.000 abstract description 21
- 208000024770 Thyroid neoplasm Diseases 0.000 abstract description 16
- 201000002510 thyroid cancer Diseases 0.000 abstract description 13
- 230000007246 mechanism Effects 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000006467 substitution reaction Methods 0.000 abstract description 6
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 230000009977 dual effect Effects 0.000 abstract description 3
- 102100022749 Aminopeptidase N Human genes 0.000 description 90
- 101150058497 ANPEP gene Proteins 0.000 description 86
- 210000004027 cell Anatomy 0.000 description 50
- 238000006243 chemical reaction Methods 0.000 description 34
- 101100481912 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) tpc-1 gene Proteins 0.000 description 21
- 206010028980 Neoplasm Diseases 0.000 description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 18
- 150000001875 compounds Chemical class 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 238000000799 fluorescence microscopy Methods 0.000 description 10
- 230000004044 response Effects 0.000 description 10
- 210000002700 urine Anatomy 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 238000001819 mass spectrum Methods 0.000 description 8
- 238000003032 molecular docking Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 231100000135 cytotoxicity Toxicity 0.000 description 6
- 230000003013 cytotoxicity Effects 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000012496 blank sample Substances 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 230000002132 lysosomal effect Effects 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 229940125904 compound 1 Drugs 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000010898 silica gel chromatography Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 230000007704 transition Effects 0.000 description 4
- 238000012800 visualization Methods 0.000 description 4
- QZDDFQLIQRYMBV-UHFFFAOYSA-N 2-[3-nitro-2-(2-nitrophenyl)-4-oxochromen-8-yl]acetic acid Chemical compound OC(=O)CC1=CC=CC(C(C=2[N+]([O-])=O)=O)=C1OC=2C1=CC=CC=C1[N+]([O-])=O QZDDFQLIQRYMBV-UHFFFAOYSA-N 0.000 description 3
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 description 3
- VGGGPCQERPFHOB-UHFFFAOYSA-N Bestatin Natural products CC(C)CC(C(O)=O)NC(=O)C(O)C(N)CC1=CC=CC=C1 VGGGPCQERPFHOB-UHFFFAOYSA-N 0.000 description 3
- 238000003775 Density Functional Theory Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 238000004770 highest occupied molecular orbital Methods 0.000 description 3
- 238000011503 in vivo imaging Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000004768 lowest unoccupied molecular orbital Methods 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000006862 quantum yield reaction Methods 0.000 description 3
- 238000004088 simulation Methods 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 210000001685 thyroid gland Anatomy 0.000 description 3
- 208000013076 thyroid tumor Diseases 0.000 description 3
- 229950009811 ubenimex Drugs 0.000 description 3
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000032798 delamination Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000012502 diagnostic product Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000012632 fluorescent imaging Methods 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Chemical compound OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- QVHJQCGUWFKTSE-YFKPBYRVSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-YFKPBYRVSA-N 0.000 description 1
- AXKGIPZJYUNAIW-UHFFFAOYSA-N (4-aminophenyl)methanol Chemical compound NC1=CC=C(CO)C=C1 AXKGIPZJYUNAIW-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 description 1
- 108090000915 Aminopeptidases Proteins 0.000 description 1
- 102000004400 Aminopeptidases Human genes 0.000 description 1
- 101001044245 Arabidopsis thaliana Insulin-degrading enzyme-like 1, peroxisomal Proteins 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 229910020366 ClO 4 Inorganic materials 0.000 description 1
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 238000004057 DFT-B3LYP calculation Methods 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- FFFHZYDWPBMWHY-UHFFFAOYSA-N HOMOCYSTEINE Chemical compound OC(=O)C(N)CCS FFFHZYDWPBMWHY-UHFFFAOYSA-N 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108010004098 Leucyl aminopeptidase Proteins 0.000 description 1
- 102000002704 Leucyl aminopeptidase Human genes 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 108030004467 Membrane alanyl aminopeptidases Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 1
- PHSPJQZRQAJPPF-UHFFFAOYSA-N N-alpha-Methylhistamine Chemical compound CNCCC1=CN=CN1 PHSPJQZRQAJPPF-UHFFFAOYSA-N 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- 102000003797 Neuropeptides Human genes 0.000 description 1
- 108090000189 Neuropeptides Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical class [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 102000016679 alpha-Glucosidases Human genes 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000002675 image-guided surgery Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000010983 kinetics study Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N methionine Chemical compound CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000004776 molecular orbital Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
- C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0028—Oxazine dyes
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6447—Fluorescence; Phosphorescence by visual observation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Materials Engineering (AREA)
- Plasma & Fusion (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用，属于荧光探针技术领域。本发明提所述荧光探针LAN‑apn的分子结构如式I所示，在制备时，将荧光团LAN‑OH与丙氨酸残基通过对氨基苯甲醇连接子连接在一起，经取代后即可构建得到一种基于ICT机理的用于生命系统中APN成像的荧光探针LAN‑apn。该探针具有稳定性高、选择性好、水溶性好、灵敏度高和检出限低等良好性能，能够通过荧光法和比色法双模式实现对APN的体外检测。本发明所述探针LAN‑apn为甲状腺癌的诊断提供了一种有效策略。
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用。
背景技术
丙氨酸氨基肽酶(APN/CD13，EC 3.4.11.2)，也被称为二聚体膜蛋白，是锌金属肽酶家族的重要成员[1]，广泛存在于哺乳动物中，并参与多种生理和病理过程，包括信号转导、神经肽降解、免疫反应和抗原处理[2]。APN因其在肿瘤侵袭、转移、血管生成等过程中发挥重要作用[3]，被发现在肝癌、乳腺癌、卵巢癌、膀胱癌等多种肿瘤内上调，可作为一种很有前景的癌症诊断标志物[4]。因此，开发一种检测、成像生命系统中APN水平的方法对与之相关癌症的诊断具有重要意义。在众多检测方法中，小分子荧光探针因其操作简便、无损、无创、高时空分辨率等明显优势[5]，已成为荧光成像和生物过程探索中最灵活的造影剂之一[6]，被广泛应用于生物信号可视化和图像引导手术中[7]。然而甲状腺癌内源性APN荧光成像的探针尚未报道。
参考文献：
[1]CHEN L,LIN Y L,PENG G,et al.Structural Basis for MultifunctionalRoles ofMammalian Aminopeptidase N[J].Proc Natl Acad Sci U S A.2012,109:17966-17971.
[2]XU Y L A W.The Structure and Main Functions ofAminopeptidase N[J].Current Medicinal Chemistry.2007,14:639-647.
[3]LIU Y,XU C,LIU H W,et al.Precipitated Fluorophore-Based MolecularProbe for in Situ Imaging ofAminopeptidase N in Living Cells and Tumors[J].Anal Chem.2021,93:6463-6471.
[4]MAWRIN C,WOLKE C,HAASE D,et al.Reduced Activity of Cd13/Aminopeptidase N(Apn)in Aggressive Meningiomas Is Associated with IncreasedLevels ofSparc[J].BrainPathol.2010,20:200-210.
[5]LI X,GAO X,SHI W,et al.Design Strategies for Water-Soluble SmallMolecular Chromogenic andFluorogenic Probes[J].Chem Rev.2014,114:590-659.
[6]LI H,KIM D,YAO Q,et al.Activity-Based Nir Enzyme FluorescentProbes for the Diagnosis of Tumors and Image-Guided Surgery[J].Angew Chem IntEd Engl.2021,60:17268-17289.
[7]LI J,KWON Y,CHUNG K S,et al.Naphthalene-Based Fluorescent Probesfor Glutathione and Their Applications in Living Cells and Patients withSepsis[J].Theranostics.2018,8:1411-1420.
发明内容
本发明的目的在于提供一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用，所述荧光探针LAN-apn基于ICT机理响应APN，实现对肿瘤细胞以及肿瘤内源性APN的荧光成像，具有稳定性高、选择性好、水溶性好、灵敏度高、检出限低等优势。
本发明提供了一种特异性识别丙氨酸氨基肽酶的荧光探针，所述荧光探针具有式I所示的结构；
本发明还提供了上述荧光探针的制备方法，包括以下步骤：
(1)将LAN-OH与式II所示化合物进行第一取代反应，得式III所示化合物；
(2)利用式III所示化合物进行第二取代反应，得式I所示荧光探针；
优选的，步骤(1)所述LAN-OH具有如式IV所示的结构；
本发明还提供了上述荧光探针在制备检测丙氨酸氨基肽酶的产品中的应用。
本发明还提供了上述荧光探针在制备疾病诊断产品中的应用，所述疾病的诊断生物标志物包括丙氨酸氨基肽酶。
优选的，所述疾病包括肿瘤。
优选的，所述疾病诊断产品为可视化诊断产品。
优选的，所述疾病诊断产品基于荧光法或比色法完成可视化诊断。
有益效果：本发明提供了一种特异性识别丙氨酸氨基肽酶的荧光探针，所述荧光探针的分子结构如式I所示，在制备时，将荧光团LAN-OH与丙氨酸残基通过对氨基苯甲醇连接子连接在一起，经取代后即可构建得到一种基于ICT机理的用于生命系统中APN成像的荧光探针LAN-apn。该探针具有稳定性高、选择性好、水溶性好、灵敏度高和检出限低等良好性能，能够通过荧光法和比色法双模式实现对APN的体外检测。此外，探针LAN-apn在人尿液中良好的加标回收率证明其能够用于尿液复杂样品中APN的精准检测。基于探针LAN-apn的低细胞毒性，本发明成功将其应用于甲状腺癌细胞和正常甲状腺细胞内源性APN成像并证实了APN在甲状腺癌细胞中明显上调。在活体成像中，探针LAN-apn能够通过响应APN而实现甲状腺肿瘤的可视化。因此，本发明所述探针LAN-apn为甲状腺癌的诊断提供了一种有效策略。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为荧光探针LAN-apn的完整合成路线图；
图2为利用荧光探针LAN-apn检测APN的检测机理示意图；
图3为溶剂DMSO-d6中化合物LAN-OH的1H-NMR谱；
图4为溶剂DMSO-d6中化合物LAN-OH的13C-NMR谱；
图5为化合物LAN-OH的LC-HRMS谱；
图6为溶剂DMSO-d6中化合物LAN-apn的1H-NMR谱；
图7为溶剂DMSO-d6中化合物LAN-apn的13C-NMR谱；
图8为化合物LAN-apn的LC-HRMS谱；
图9为探针LAN-apn的光谱特性结果图；(A)和(B)为探针(10μM)和反应体系(10μM探针与1000ng/mLAPN)的荧光光谱/吸收光谱；(C)为探针(10μM)和反应体系(10μM探针与1000ng/mLAPN)在连续580nm激发光照射下的光稳定性结果图；(D)为探针(10μM)和反应体系(10μM探针与1000ng/mLAPN)在25～42℃温度内的荧光变化图；(E)为10μM探针与不同浓度APN反应时间变化图；(F)为pH对探针(10μM)和反应体系(10μM探针与1000ng/mLAPN)的影响结果图；
图10为探针LAN-apn的分析性能结果图；(A)为10μM探针LAN-apn与不同浓度APN(0～1000ng/mL)响应的荧光光谱；(B)为反应体系在642nm处的荧光强度与APN浓度(0～400ng/mL)之间的线性关系；(C)为10μM探针LAN-apn与不同浓度APN(0～1000ng/mL)响应的吸收光谱；(D)为反应体系在575nm处的吸光度与APN浓度(0～400ng/mL)之间的线性关系；
图11为探针LAN-apn的酶反应动力学研究结果图；(A)为270ng/mLAPN与不同浓度(1，2，3，4，5，6，7，8，9，10μΜ)探针LAN-apn酶促反应的Michaelis-Menten图；(B)为酶促反应的Lineweaver-bulk图；
图12为在各种分析物存在时(除非另有说明否则均为1mM)10μM探针LAN-apn的荧光强度：1.Blank；2.Mn2+；3.Li+；4.Cu2+；5.Mg2+；6.Ag+；7.Hg2+；8.Ca2+；9.Cd2+；10.Pb2+；11.Ni2 +；12.Al3+；13.Zn2+；14.K+；15.Ba2+；16.Fe2+；17.Co2+；18.Na+；19.Fe3+；20.AcO-；21.CN-；22.ClO4-；23.HSO4-；24.SCN-；25.CN-；26.H2PO4-；27.F-；28.Cl-；29.Br-；30.I-；31.Lysine；32.Valine；33.Tryptophan；34.Aspartic acid；35.Histidine；36.L-Arginine；37.L-Alanine；38.DL-Homocysteine；39.DL-Phenylalanine；40.L-Cysteine；41.L-Leucine；42.DL-Methionine；43.Glycine；44.L-Glutamic；45.D(+)-Mannose；46.D-Fructose；47.Dithiothreitol；48.L-Ascorbic；49.Chitosan；50.Citrulline；51.5000U/LAlkaline Phosphatase；52.1μM Thrombin；53.10mg/L Glucose Oxidase；54.10mg/LGlucose Oxidase；55.10mg/L Carcinoembryonic Antigen；56.10mg/LAlbumin Bovine V；57.20mg/L Hyaluronidase；58.7000U/L Maltase；59.H2O2；60.400ng/mL APN；
图13为荧光团LAN-OH的质谱；
图14为反应体系的质谱；
图15为探针LAN-apn(10μM)，荧光团LAN-OH(10μM)，反应体系(10μM探针与400ng/mLAPN)的HPLC分析结果图；
图16为抑制剂Bestatin对探针LAN-apn(10μM)、荧光团LAN-OH(10μM)及反应体系(10μM探针与400ng/mLAPN)荧光强度的影响结果图；
图17为探针LAN-apn与APN的分子对接模拟及探针LAN-apn与荧光团LAN-OH的HOMO/LUMO能级结果图；
图18为不同浓度探针LAN-apn的细胞毒性分析结果图；
图19为细胞内LAN-apn荧光强度随时间的变化结果图；(A)为Tpc-1细胞内荧光强度随时间变化趋势；(B)为对应(A)中Tpc-1细胞内平均荧光强度值；
图20为探针LAN-apn应用对Tpc-1甲状腺癌细胞和Nthy-oris-1正常甲状腺细胞内源性APN的荧光成像；(A)为Tpc-1细胞和Nthy-oris-1细胞内源APN荧光成像；(B)为对应(A)中细胞内平均荧光强度值；
图21为使用探针LAN-apn和商用溶酶体染料Lyso-Tracker-Green进行共定位成像结果图；(A)为溶酶体染料Lyso-Tracker-Green的绿色荧光通道成像(λex＝488nm，λem＝500-550nm)；(B)为探针LAN-βgal的红色荧光通道成像(λex＝559nm；λem＝580-680nm)；(C)为绿色和红色通道的合并图像；(D)为明场；(E)为Pearson系数；(F)为对应(C)中短线处荧光强度强度分布图；
图22为(A)正常小鼠、(B)荷瘤小鼠(Tpc-1肿瘤)注射探针LAN-apn(200μM，50μL)后的荧光成像效果图；
图23为探针LAN-apn孵育后活体组织的成像结果图。
具体实施方式
本发明提供了一种特异性识别丙氨酸氨基肽酶的荧光探针，所述荧光探针具有式I所示的结构；
本发明所述荧光探针LAN-apn可通过荧光法和比色法有效地检测APN，如在荧光光谱中，APN的加入使探针LAN-apn在以642nm为中心的发射峰显著增强(λex＝580nm)并伴随着溶液颜色由黄色向荧光红的转变(365nm紫外灯照射下)；在吸收光谱中，APN的加入使探针LAN-apn在以575nm为中心的吸收峰显著增强并伴随着溶液颜色由淡黄色向红色的显著转变。且所述荧光探针LAN-apn具有高稳定性，如良好的光稳定性、25～42℃范围内良好的热稳定性、pH 5～9范围内良好的稳定性，细胞毒性极低，可应用于活体检测。
本发明还提供了上述荧光探针的制备方法，包括以下步骤：
(1)将LAN-OH与式II所示化合物进行第一取代反应，得式III所示化合物；
(2)利用式III所示化合物进行第二取代反应，得式I所示荧光探针；
本发明所述荧光探针LAN-apn的完整合成路线优选如图1所示，其中化合物1的合成方法优选参照ZHANG M等进行(ZHANG M,TIAN Z,WANG J,et al.VisualAnalysis andInhibitor Screening ofLeucineAminopeptidase,a KeyVirulence Factor forPathogenic Bacteria-Associated Infection[J].ACS Sens.2021,6:3604-3610.)；化合物2(式II)的合成方法优选参照CHAIY等进行(CHAIY,GAO Y,XIONG H,et al.A near-Infrared Fluorescent Probe for Monitoring Leucine Aminopeptidase in LivingCells[J].Analyst.2019,144:463-467.)。本发明所述LAN-OH优选具有如式IV所示的结构，且对其制备方法并没有特殊限定，参考本领域的常规文献所记载的方法进行制备即可。
本发明还提供了上述荧光探针在制备检测丙氨酸氨基肽酶的产品中的应用。
本发明所述荧光探针LAN-apn对APN具有良好的亲和力、高度的敏感性和高度特异性，且对APN的检测机理优选如图2所示：APN特异性识别并切断探针LAN-apn的丙氨酸残基，随后对氨基苯甲醇连接子自发离去最终释放出荧光团LAN-OH从而实现探针LAN-apn对APN的检测。
本发明还提供了上述荧光探针在制备疾病诊断产品中的应用，所述疾病的诊断生物标志物包括丙氨酸氨基肽酶。
本发明实施例中证实了，利用浓度达到50μM的荧光探针LAN-apn影响细胞，结果细胞存活率仍高于90％，说明探针LAN-apn对甲状腺癌细胞低细胞毒性，可应用于活体检测。本发明所述荧光探针LAN-apn在细胞内具有良好的稳定性，且可定位于细胞的溶酶体部位，APN在甲状腺癌细胞内过表达并且探针LAN-apn能够通过与APN的响应实现细胞内源性APN的可视化。本发明所述可视化优选包括可通过荧光法或比色法完成可视化诊断。因此，利用本发明所述荧光探针LAN-apn具有稳定性高、选择性好、水溶性好、灵敏度高、检出限低等优势，可通过荧光法和比色法双模式检测APN，并能够用于活体组织体外成像以评估APN水平。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
本发明实施例中，除非另有说明，否则本工作中使用的所有化学试剂均为购买自药品供应商的分析试剂级，使用时未经进一步提纯。1H谱和13C谱数据通过Avance III500NMR波谱仪(Bruker Inc.，德国)获得，质谱数据通过TSQ QuantumAccess MAX三重四极杆质谱仪(Thermo Fisher Scientific，美国)获得。丙氨酸氨基肽酶购买自Sigma-Aldrich。Lyso-Tracker-Green购买自碧云天生物。Tpc-1细胞、Nthy-oris-1细胞购买自赛百慷(上海)生物技术股份有限公司。BALB/c-nu小鼠购买自北京华阜康生物科技股份有限公司。所有水溶液均由所有溶液均采用超纯水配制(Milli-Q system,Millipore，美国)。
实施例1
根据图1所示路线合成探针LAN-apn：
(1)化合物1的合成
冰浴条件下，将4-氨基苯甲醇(400mg，3.25mmol)、N-叔丁氧羰基-L-丙氨酸(567mg，3mmol)和2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.14g，3mmol)溶解在15mL无水四氢呋喃中，在氮气保护下搅拌10min。然后滴加N,N-二异丙基乙胺(523μL，3mmol)继续搅拌20min。最后在室温条件下搅拌过夜。以石油醚和乙酸乙酯的混合物(v/v＝1：1)为流动相，经硅胶柱层析法纯化得到乳白色固体化合物1(产率72％)。
(2)化合物2(式II)的合成
冰浴条件下，将化合物1(0.294g，1mmol)溶于10mL无水四氢呋喃中搅拌，10min后滴加三溴化磷(142μL，1.5mmol)并继续搅拌3h。停止反应，向其中滴加饱和碳酸钠溶液使其调整为中性，然后加入二氯甲烷进行萃取，分层后将二氯甲烷层低温旋蒸得乳白色固体粗产物(产率60％)，无需进一步提纯直接用于下一步反应。
3)化合物3的合成
冰浴条件下，依次向烧杯中加入350mL冰水和6.3mL浓硫酸并充分搅拌，然后将间苯二酚(14.5g，0.13mol)与亚硝酸钠(10.8g，0.15mol)的混合液逐滴加入烧杯，滴加完毕后继续搅拌20min后停止反应，然后将产物进行抽滤、冷冻干燥。最终产物为明黄色固体，不需要进一步提纯即可用于下一步合成(17.93g，96％)。
(4)化合物LAN-OH(式IV)的合成
室温下，将化合物3(0.153g，1.1mmol)和1-萘酚(0.144g，1mmol)加入到5mL二氯甲烷中进行充分搅拌。随后将3mL浓盐酸逐滴加入反应瓶，搅拌5h后停止反应。以石油醚和乙酸乙酯(v/v＝5:1)作为流动相，通过硅胶柱层析法进行纯化，化合物LAN-OH为黄褐色固体(130mg，65％)。1HNMR(300MHz,DMSO-d6)δ8.60(d,J＝8.0Hz,1H),8.14(d,J＝7.8Hz,1H),7.85(t,J＝7.5Hz,1H),7.78(d,J＝7.4Hz,1H),7.70(d,J＝8.7Hz,1H),6.85(d,J＝8.8Hz,1H),6.76(s,1H),6.36(s,1H)(图3)。13C NMR(75MHz,DMSO-d6)δ182.42,161.50,151.35,145.32,142.41,132.03,131.33,131.20,131.09,131.01,126.14,125.11,123.82,114.02,105.70,105.64,101.77(图4)。MS(LC-HRMS,m/z)for C16H10NO3 +[M+H]+:calculated,264.0655；found:264.0652(图5)。
(4)化合物4(式III)的合成
室温条件下，向反应瓶中相继加入化合物LAN-OH(0.263g，1mmol)、化合物2(0.356g，1mmol)和碳酸钾(0.138g，1mmol)，最后加入10mLN,N-二甲基甲酰胺搅拌3h。停止反应，向反应瓶中加入饱和食盐水，将产生的沉淀离心后进一步通过硅胶柱层析法(二氯甲烷:甲醇＝30:1)进一步纯化得黄色固体化合物4(产率73％)。
(5)探针LAN-apn(式I)的合成
室温条件下，将化合物4(0.24g，0.446mmol)溶解于5mL二氯甲烷中，然后滴加1mL三氟乙酸搅拌1h。停止反应，向反应瓶中加入饱和食盐水，分层后收集有机层进行旋蒸。最后通过硅胶柱层析法(二氯甲烷:甲醇＝20:1)进一步纯化，得到黄色固体产物(产率80％)。1H NMR(500MHz,DMSO-d6)δ10.85(s,1H),8.56(d,J＝7.6Hz,1H),8.11(d,J＝7.8Hz,1H),7.96(s,1H),7.85(t,J＝6.9Hz,1H),7.78(dd,J＝15.0,8.1Hz,2H),7.71(d,J＝8.5Hz,2H),7.48(d,J＝8.5Hz,2H),7.14(d,J＝2.6Hz,1H),7.07(dd,J＝8.8,2.6Hz,1H),6.36(s,1H),5.20(s,2H),4.05(d,J＝7.0Hz,1H),1.46(d,J＝7.0Hz,4H)(图6)。13C NMR(126MHz,DMSO-d6)δ182.51(s),168.85(s),162.33(s),161.28(s),151.28(s),145.13(s),143.63(s),138.35(s),132.15(s),131.48(s),131.39(s),131.07(s),130.64(s),128.81(s),127.02(s),125.16(s),123.99(s),119.40(s),113.76(s),105.91(s),101.26(s),69.94(s),49.11(s),35.80(s),30.79(s),17.52(s)(图7)。MS(m/z)for C26H22N3O4 +[M+H]+:calculated,440.1610；found:440.1618(图8)。
实施例2
1、光谱检测过程
将探针LAN-apn溶解于二甲基亚砜(DMSO)制得1mM原液。由10μL探针原液、一定体积浓度的待分析物和一定体积的PBS构成的1mL测试样品溶液分别通过荧光光谱仪和紫外可见分光光度计采集数据。除非特殊说明，否则待测体系在37℃含有1％(v/v)DMSO的PBS缓冲溶液(10mM，pH 7.4)中反应70min后用于检测。使用荧光光谱仪测试时，激发光设置为580nm，狭缝为2.5/2.5nm，电压为700V，记录590-750nm范围的发射光谱。使用紫外可见分光光度计测试时，使用1cm石英比色皿记录450-650nm范围的吸收光谱。
结果如图9所示，在荧光光谱中，APN的加入使探针LAN-apn在以642nm为中心的发射峰显著增强(λex＝580nm)并伴随着溶液颜色由黄色向荧光红的转变(365nm紫外灯照射下，图9中A)。在吸收光谱中，APN的加入使探针LAN-apn在以575nm为中心的吸收峰显著增强并伴随着溶液颜色由淡黄色向红色的显著转变(图9中B)。以上结果表明探针LAN-apn可以通过荧光法和比色法有效地检测APN。
本发明测试了探针LAN-apn和反应体系的光稳定性。如图9中C所示，在580nm激发光连续80min的照射下，无论探针LAN-apn还是反应体系的荧光强度均无明显的变化，这表明其良好的光稳定性。随后，测试温度对探针LAN-apn和反应体系的影响。结果如图9中D所示，在25～42℃温度范围内，探针LAN-apn的荧光强度基本保持不变，证明探针在25～42℃内良好的热稳定性；而反应体系荧光强度随着温度的升高而逐渐增强，推测适当范围内温度的升高加快了酶促反应。
本发明探究探针LAN-apn与APN的响应情况随时间的变化情规律，如图9中E所示，探针LAN-apn与一系列浓度APN响应的荧光强度均随时间的进行而逐渐增强，具体表现为在前70min内增强较快而70min后速度明显减慢。最后，探讨pH对LAN-apn的影响。如图9中F所示，在pH 5-9范围内，探针LAN-apn荧光强度基本保持不变，证明探针LAN-apn对pH的稳定性；而反应体系荧光强度首先随着pH的增大而逐渐增强，在pH 7.4处达到最大值后开始逐渐减弱，这可能是因为APN在酸性和碱性环境中活性较差导致的。综合以上实验结果并考虑到生理条件，后续体外实验均在37℃下的PBS(10mM，pH 7.4)中响应70min后用于检测。
2、检出限及荧光量子产率的测定
(1)检出限的测定。通过荧光法和比色法分别采集20个10μM探针LAN-apn空白样品在642nm处的荧光强度和575nm处的吸光度以计算出标准偏差σ。根据公式DL＝3σ/k计算出探针LAN-apn对APN的检出限，其中：σ：空白样品的标准偏差；k：标准曲线斜率；DL：检出限。
在最佳实验条件下，利用探针LAN-apn对APN进行定量检测。结果如图10所示，在荧光法定量检测APN中(图10中A)，随着APN浓度逐渐增大(0～1000ng/mL)反应体系荧光逐渐增强(λex＝580nm，λemmax＝642nm)，反应体系荧光强度与APN浓度(0～400ng/mL)之间存在良好的线性关系(r＝0.9969)，检出限(DL)低至0.069ng/mL(图10中B)。探针LAN-apn对APN的检测在荧光增强倍数、激发/发射波长及检出限等方面与其他已报道的荧光探针的比较如表1所示，探针LAN-apn表现出明显的检测优势。在比色法定量检测APN中(图10中C)，随着APN浓度逐渐增大(0～1000ng/mL)反应体系在575nm处的吸光度逐渐增强，吸光度与APN浓度(0～400ng/mL)之间存在良好的线性关系(r＝0.9892)，检出限(DL)低至4.5ng/mL(图10中D)。因此探针LAN-apn能够通过荧光法和比色法定量准确检测APN。
表1探针LAN-apn与其它已报道的荧光探针分析性能的比较
表1中各探针除LAN-lap外，其余探针的来源依次为：
HE X,HU Y,SHI W,et al.Design,Synthesis and Application ofanear-Infrared Fluorescent Probe for in Vivo Imaging of Aminopeptidase N[J].ChemCommun(Camb).2017,53:9438-9441.
LIU Y,XU C,LIU H W,et al.Precipitated Fluorophore-Based MolecularProbe for in Situ Imaging ofAminopeptidase N in Living Cells and Tumors[J].Anal Chem.2021,93:6463-6471.
HE X,XU Y,SHI W,et al.Ultrasensitive Detection of AminopeptidaseNActivity in Urine and Cells with a Ratiometric Fluorescence Probe[J].AnalChem.2017,89:3217-3221.
LIU S-Y,WANG H,ZOU X,et al.De Novo Design of an UltrasensitiveFluorogenic Probe for Aminopeptidase N Sensing in Living System[J].Sensorsand Actuators B:Chemical.2022,363
LI H,LI Y,YAO Q,et al.In Situ Imaging of Aminopeptidase N Activity inHepatocellular Carcinoma:A Migration Model for Tumour Using an ActivatableTwo-PhotonNirFluorescent Probe[J].Chem Sci.2019,10:1619-1625.
(2)荧光量子产率的测定。首先制备待测样品和空白样品。10μM探针LAN-apn待测样品：10μL探针原液加入到990μL PBS中(10mM，pH 7.4)；10μM荧光团LAN-OH待测样品：10μL荧光团(1mM)加入到990μL PBS中(10mM，pH 7.4)；空白样品：1mL PBS(10mM，pH 7.4)。待测样品及空白样品的荧光量子产率由稳态瞬态荧光光谱仪(FLS920，Edinburgh Instrument)测得(λex＝580nm)。
3、酶动力学实验
首先在PBS(10mM，pH 7.4)中制备不同浓度的探针LAN-apn(1，2，3，4，5，6，7，8，9，10μM)，然后分别加入270ng/mL的APN，立即在10min范围内每1min记录一次荧光强度(λex＝580nm，λemmax＝642nm)，初始反应速率由线性响应范围内的数据确定，根据公式(Michaelis-Menten equation)和(Lineweaver-Burk equation)计算出水解过程动力学参数。
水解过程动力学参数由下式计算:
Michaelis-Menten equation:
Lineweaver-Burk equation:
其中，v：初始反应速率；V：初始反应速率最大值；[S]：底物(探针)浓度；Km：Michaelis常数。
利用Michaelis-Menten方程和Lineweaver-Burk双倒数作图法探究探针LAN-apn与APN之间的反应动力学。在Michaelis-Menten图中(图11中A)，随着探针LAN-apn浓度的逐渐增大，反应初速度呈现出由快速到缓慢的增强效果，我们根据初速度和探针LAN-apn浓度经换算作出Lineweaver-Burk图(图11中B)，计算出最大速度Vmax＝82.58μM min-1，米氏常数Km＝5.85μM，这表明探针LAN-apn对APN具有良好的亲和力和高度的敏感性。
为了评估探针LAN-apn对APN的选择性，本发明探究了包括阳离子、阴离子、小分子、蛋白质和酶等一系列环境及生命系统中常见的潜在干扰物对探针LAN-apn的干扰情况。如图12所示，即使干扰物的浓度远远高于分析物APN，探针LAN-apn依旧对除APN以外的其他干扰物基本无响应，且探针LAN-apn与APN响应的荧光强度远远高于其他干扰物，这表明探针LAN-apn对APN具有高选择性，这为探针LAN-apn在复杂环境中屏蔽来自其他干扰物的影响而准确检测APN提供了重要保障。
4、尿液中APN的加标回收率
首先将新鲜的人尿液样品离心(8000rpm，5min)，取900μL上清液，向其中依次加入90μL不同浓度的APN溶液(最终浓度分别为20，40，60ng/mL)和10μL探针LAN-apn原液。最后，在37℃下孵育70min后用于荧光测试。
结果如表2所示，尿液中加入标准浓度20，40，60ng/mL APN的回收率范围在93.4～101.4％，表明探针LAN-apn在人尿液中具有良好的加标回收率，证明探针LAN-apn能够用于人尿中APN的精准检测。
表2人尿液中APN的加标回收率(n＝3)
a表示平均值±标准差。
5、分子对接与理论计算
(1)利用Autodock 4.0软件对探针LAN-apn与APN进行分子对接模拟(PDB code:4fkh)并使用Autodock Tools对APN结构进行处理。在对接探针LAN-apn之前，分配了AD4原子类型并计算了Gasteiger电荷。所有可旋转键均设置为主动扭转。对接周期、探针LAN-apn和APN参数均按缺省值配置。通过PyMOL 2.3.3和Discovery Studio 2020软件对对接模型进行可视化。
(2)使用Gaussian 16软件在B3LYP(GD3BJ)/def2-SVP水平上，利用密度泛函理论(DFT)和时变DFT(TDDFT)对探针LAN-βgal的最佳几何结构和电子结构进行了计算并利用Multiwfn 3.7软件对所得数据进行进一步分析。
为了探究探针LAN-apn与APN的响应机理，本发明进行了质谱检测，HPLC分析和抑制剂实验。在质谱检测中，探针LAN-apn的质谱峰m/z为440.1236(图8)，荧光团LAN-OH的质谱峰m/z为264.0667(图13)，而反应体系出现m/z为264.0657的质谱峰(图14)，这与荧光团LAN-OH的质谱峰相吻合。
HPLC分析结果如图15所示，探针LAN-apn出峰时间为1.60min，荧光团LAN-OH出峰时间为2.52min，而反应体系的出峰时间为1.60min和2.52min，其中2.52min新出现的峰与荧光团LAN-OH的出峰时间相吻合。
抑制剂实验结果如图16所示，抑制剂Bestatin对探针LAN-apn和荧光团LAN-OH的荧光强度基本无影响，而明显抑制了反应体系的荧光强度。综合以上实验的结果，推测探针LAN-apn与APN的响应机理如图2所示，即APN特异性识别并切断探针LAN-apn的丙氨酸残基，随后对氨基苯甲醇连接子自发离去最终释放出荧光团LAN-OH从而实现探针LAN-apn对APN的检测。
对探针LAN-apn与APN的分子对接方式进行模拟，结果如图17所示，探针LAN-apn与APN中的4个氨基酸(Pro432，Gln693，Tyr689，Ser697)形成5个氢键，结合能为-10.4kcal/mol，表明探针LAN-apn与APN具有较强的结合亲和力。继续对探针LAN-apn和荧光团LAN-OH的分子轨道进行理论计算。探针LAN-apn的HOMO和LUMO都主要位于整个荧光团中，虽然在荧光团LAN-OH的HOMO和LUMO上没有电子密度的再分布，但对应典型ICT的S1到S0的跃迁(625.7nm,f＝1.1193)导致探针LAN-apn与APN响应后产生强烈的荧光。荧光团LAN-OH(2.58eV)的能隙低于探针LAN-apn(2.99eV)，可以合理地证明APN的加入使探针LAN-apn的吸收波长的红移。以上分子对接模拟和理论计算的结果支持图2中所提出的探针LAN-apn与APN的传感机理。
实施例3
3.1细胞实验
3.1.1细胞培养
所有细胞均在含有10％胎牛血清(FBS)、100U/mL青霉素和100μg/mL链霉素的RPMI1640中培养并保存在37℃的含5％二氧化碳/95％空气的培养箱中。
3.1.2细胞毒性分析
通过MTT法进行毒性分析。向96孔板每孔中加入100μL甲状腺癌细胞悬液(105个细胞/mL)，将其保存在37℃含5％二氧化碳/95％空气的培养箱中12h。然后在每孔中加入10μL不同浓度的探针LAN-apn溶液(5，10，20，30，50μM)并孵育24h。接下来在每孔加入20μLMTT溶液，4h后除去MTT溶液并向每孔加入150μL DMSO以溶解甲瓒晶体。最后使用酶标仪测定细胞存活率。
使用CCK-8方法评估探针LAN-apn的细胞毒性，结果如图18所示，即使探针LAN-apn浓度高达50μM，细胞存活率仍高于90％，说明探针LAN-apn对甲状腺癌细胞低细胞毒性。
3.1.3细胞成像
(1)时间成像实验。准备9组Tpc-1细胞并分别用10μM探针LAN-apn孵育，并记录不同时间时刻细胞的成像情况。
基于低细胞毒性，探针LAN-apn被进一步应用于细胞成像。首先探究了细胞内荧光强度随时间的变化规律。如图19中A、B所示，Tpc-1细胞内荧光强度随着时间的进行逐渐增强，大约在60min时达到最大值且至少在2h内荧光保持不变，表明探针LAN-apn在细胞中良好的稳定性。
(2)细胞内源性APN成像。第一组将Tpc-1细胞(甲状腺癌细胞)和Nthy-oris-1细胞(正常甲状腺细胞)作为空白对照组。第二组Tpc-1细胞和Nthy-oris-1细胞分别用10μM探针LAN-apn孵育1h后进行荧光成像。第三组Tpc-1细胞和Nthy-oris-1细胞先用100μMBestatin(APN的一种抑制剂)孵育1h后用1mLPBS(10mM，pH 7.4)洗涤3次，再分别用10μM探针LAN-apn孵育1h，最后进行荧光成像。
结果如图20中A、B所示，Tpc-1细胞显示出显著的荧光信号而Nthy-oris-1细胞显示微弱的荧光信号且抑制剂Bestatin的加入抑制了细胞内的荧光信号，尤其是Tpc-1细胞。以上结果表明APN在甲状腺癌细胞内过表达并且探针LAN-apn能够通过与APN的响应实现细胞内源性APN的可视化。
(3)共定位成像实验。Tpc-1细胞用溶酶体染料Lyso-Tracker-Green(50nM)孵育30min后用1mLPBS(10mM，pH 7.4)洗涤3次，再分别用10μM探针LAN-apn孵育1h后用于荧光成像。
通过将探针LAN-apn与商用溶酶体染料Lyso-Tracker-Green共孵育Tpc-1细胞对探针LAN-apn在细胞内成像位置进行探究。结果如图21所示，溶酶体染料Lyso-Tracker-Green的绿色通道(图21中A)和探针LAN-apn的红色通道(图21中B)很好的重合(图21中C)，Pearson系数高达0.85(图21中E)。以上结果表明探针定位于细胞的溶酶体部位。
3.2活体实验
3.2.1小鼠模型的建立
动物实验程序由吉林大学动物实验伦理委员会批准。将1×107Tpc-1甲状腺癌细胞悬浮于300μL PBS(10mM，pH 7.4)中，然后将Tpc-1细胞悬液接种于裸鼠背部右侧皮下以构建荷瘤小鼠模型。20天后获得肿瘤模型。活体成像实验在IVIS Lumina LT Series III小动物成像系统上进行，激发波长为560nm，滤光片为650nm。
3.2.2活体成像
(1)对比实验。将小鼠分为对照组、肿瘤组(Tpc-1肿瘤)两组，其中肿瘤组小鼠瘤内注射探针LAN-apn(200μM，50μL)，1h后用于荧光成像。对照组小鼠在对应位置注射探针LAN-apn(200μM，50μL)，1h后用于荧光成像。
结果如图22所示，与对照组(图22中A)相比，肿瘤组(图22中B)小鼠瘤内显示出显著的荧光信号，说明APN在甲状腺肿瘤内过表达，这与细胞成像中得到的结果相吻合(图20)。以上结果表明探针LAN-apn能够实现甲状腺肿瘤通过响应内APN的可视化。
(2)活体组织体外成像。将荷瘤小鼠(Tpc-1肿瘤)解剖后取出心、肝、脾、肺、肾、肌肉组织以及肿瘤组织，使用探针LAN-apn(200μM)进行孵育，1h后用于荧光成像。
结果如图23所示，心、脾、肺、肾、肌肉组织显示出微弱的荧光信号，而肝脏和Tpc-1肿瘤显示出明显的荧光信号。以上结果表明在心、脾、肺、肾、肌肉组织APN水平较低，而在肝脏和Tpc-1肿瘤中APN水平表达较高，探针LAN-apn能够很好地对活体组织内APN水平进行分析。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (1)
1.特异性识别丙氨酸氨基肽酶的荧光探针在制备检测丙氨酸氨基肽酶的产品中的应用，所述产品基于荧光法或比色法完成可视化诊断，所述荧光探针具有式I所示的结构；
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310415928.5A CN116462639B (zh) | 2023-04-18 | 2023-04-18 | 一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310415928.5A CN116462639B (zh) | 2023-04-18 | 2023-04-18 | 一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116462639A CN116462639A (zh) | 2023-07-21 |
| CN116462639B true CN116462639B (zh) | 2023-12-08 |
Family
ID=87183800
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310415928.5A Active CN116462639B (zh) | 2023-04-18 | 2023-04-18 | 一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116462639B (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603695A (zh) * | 2012-02-10 | 2012-07-25 | 山东大学 | 一种氨基酸-荧光团类化合物及其应用 |
| CN114315791A (zh) * | 2021-12-22 | 2022-04-12 | 东南大学 | 用于实现手术导航和微小转移瘤成像的小分子化学发光探针及其制备方法和用途 |
| CN114380786A (zh) * | 2021-12-23 | 2022-04-22 | 苏州大学 | 基于氨基肽酶激活型化学发光探针及其在恶性肿瘤的活体检测与手术导航方面的应用 |
| CN114605343A (zh) * | 2022-03-14 | 2022-06-10 | 吉林大学 | 一种荧光基团LAN-OH、荧光传感器LAN-βgal及其制备方法和应用 |
| CN114835722A (zh) * | 2022-05-12 | 2022-08-02 | 大连理工大学 | 一种用于测定农药残留的丙氨酸氨基肽酶荧光探针的合成与应用 |
-
2023
- 2023-04-18 CN CN202310415928.5A patent/CN116462639B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603695A (zh) * | 2012-02-10 | 2012-07-25 | 山东大学 | 一种氨基酸-荧光团类化合物及其应用 |
| CN114315791A (zh) * | 2021-12-22 | 2022-04-12 | 东南大学 | 用于实现手术导航和微小转移瘤成像的小分子化学发光探针及其制备方法和用途 |
| CN114380786A (zh) * | 2021-12-23 | 2022-04-22 | 苏州大学 | 基于氨基肽酶激活型化学发光探针及其在恶性肿瘤的活体检测与手术导航方面的应用 |
| CN114605343A (zh) * | 2022-03-14 | 2022-06-10 | 吉林大学 | 一种荧光基团LAN-OH、荧光传感器LAN-βgal及其制备方法和应用 |
| CN114835722A (zh) * | 2022-05-12 | 2022-08-02 | 大连理工大学 | 一种用于测定农药残留的丙氨酸氨基肽酶荧光探针的合成与应用 |
Non-Patent Citations (3)
| Title |
|---|
| An APN-activated NIR photosensitizer for cancer photodynamic therapy and fluorescence imaging;Xiao Zhou 等;Biomaterials;第253卷;第120089页 * |
| Multiplex Optical Urinalysis for Early Detection of Drug-Induced Kidney Injury;Penghui Cheng 等;Analytical chemistry;第92卷(第8期);第6166-6172页 * |
| Zhang Ming 等.Visual Analysis and Inhibitor Screening of Leucine Aminopeptidase,a Key Virulence Factor for Pathogenic Bacteria-Associated Infection.ACS sensors.2021,第6卷(第10期),Supporting Information. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116462639A (zh) | 2023-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Recent advances in multifunctional fluorescent probes for viscosity and analytes | |
| CN111499604B (zh) | 一种溶酶体靶向的Cys近红外荧光探针及其制备方法和应用 | |
| Lee et al. | Near-infrared fluorescent probes for the detection of glutathione and their application in the fluorescence imaging of living cells and tumor-bearing mice | |
| Tang et al. | A mitochondria-targetable far-red emissive fluorescence probe for highly selective detection of cysteine with a large Stokes shift | |
| Liu et al. | Recent advances in Golgi-targeted small-molecule fluorescent probes | |
| CN110283583B (zh) | γ-谷氨酰转肽酶响应型分子探针及其应用 | |
| CN109053802B (zh) | 一种比率型近红外荧光探针及其合成方法与应用 | |
| Li et al. | 1, 8-Naphthalimide-based dual-response fluorescent probe for highly discriminating detection of cys and H2S | |
| Liu et al. | Lysosome-targeted near-infrared fluorescent dye and its application in designing of probe for sensitive detection of cysteine in living cells | |
| CN113234014B (zh) | 一种检测氨肽酶n的聚集诱导发射荧光探针及其制备 | |
| Guo et al. | Fabrication of a water-soluble near-infrared fluorescent probe for selective detection and imaging of dipeptidyl peptidase IV in biological systems | |
| Han et al. | Rational design of a lysosomal-targeted ratiometric two-photon fluorescent probe for imaging hydrogen polysulfides in live cells | |
| CN110526908B (zh) | 基于2-苯乙烯基吲哚盐类衍生物长波发射可区分检测Cys/Hcy荧光探针及其应用 | |
| CN114605343B (zh) | 一种荧光基团LAN-OH、荧光传感器LAN-βgal及其制备方法和应用 | |
| Rong et al. | Double-channel based fluorescent probe for differentiating GSH and H2Sn (n> 1) via a single-wavelength excitation with long-wavelength emission | |
| CN111548790A (zh) | 一种近红外比率型荧光探针及其合成方法与应用 | |
| CN114539222A (zh) | 一种七甲川菁-萘酰亚胺杂合体的合成和作为近红外荧光探针在检测谷胱甘肽中的应用 | |
| CN116535364A (zh) | 一种特异性识别亮氨酸氨基肽酶的荧光探针及制备方法和应用 | |
| CN113637048B (zh) | 一种γ-谷氨酰转肽酶的双光子荧光探针及其制备方法和应用 | |
| Yang et al. | A near-infrared fluorescent probe for the discrimination of cysteine in pure aqueous solution and imaging of cysteine in hepatocellular carcinoma cells with facile cell-compatible ability | |
| Zhao et al. | A novel near-infrared fluorescent probe for intracellular detection of cysteine | |
| CN117603203A (zh) | 一种比率荧光探针及其制备方法和应用 | |
| CN111233928B (zh) | 一种香豆素衍生物Mito-Cys及其制备方法和应用 | |
| CN116462639B (zh) | 一种特异性识别丙氨酸氨基肽酶的荧光探针及制备方法和应用 | |
| Liao et al. | An effective HBT/indanone-based fluorescent probe for cysteine detection in cells |
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 |