WO2013174262A1 - 诊断癌症的方法、试剂盒和系统 - Google Patents
诊断癌症的方法、试剂盒和系统 Download PDFInfo
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- WO2013174262A1 WO2013174262A1 PCT/CN2013/076071 CN2013076071W WO2013174262A1 WO 2013174262 A1 WO2013174262 A1 WO 2013174262A1 CN 2013076071 W CN2013076071 W CN 2013076071W WO 2013174262 A1 WO2013174262 A1 WO 2013174262A1
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- Prior art keywords
- buffer
- cells
- cancer
- butyl
- kit
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Links
- 238000000034 method Methods 0.000 title claims abstract description 64
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 34
- 201000011510 cancer Diseases 0.000 title claims abstract description 24
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 11
- 238000010186 staining Methods 0.000 claims abstract description 83
- 239000006285 cell suspension Substances 0.000 claims abstract description 46
- 150000001875 compounds Chemical class 0.000 claims abstract description 39
- 230000003902 lesion Effects 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims abstract description 4
- 239000000872 buffer Substances 0.000 claims description 40
- 208000020816 lung neoplasm Diseases 0.000 claims description 32
- 125000000217 alkyl group Chemical group 0.000 claims description 28
- 206010006187 Breast cancer Diseases 0.000 claims description 25
- 208000026310 Breast neoplasm Diseases 0.000 claims description 25
- 206010009944 Colon cancer Diseases 0.000 claims description 25
- 208000029742 colonic neoplasm Diseases 0.000 claims description 25
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 22
- 201000005202 lung cancer Diseases 0.000 claims description 22
- -1 halogen anion Chemical class 0.000 claims description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Natural products CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 16
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 claims description 14
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 12
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 claims description 12
- 125000004491 isohexyl group Chemical group C(CCC(C)C)* 0.000 claims description 12
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 claims description 12
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 12
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 12
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 12
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 claims description 12
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 12
- 239000008363 phosphate buffer Substances 0.000 claims description 12
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 12
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 11
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 claims description 11
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- CSVGEMRSDNSWRF-UHFFFAOYSA-L disodium;dihydrogen phosphate Chemical group [Na+].[Na+].OP(O)([O-])=O.OP(O)([O-])=O CSVGEMRSDNSWRF-UHFFFAOYSA-L 0.000 claims description 10
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 9
- 150000001721 carbon Chemical group 0.000 claims description 8
- 229960002319 barbital Drugs 0.000 claims description 7
- 125000004432 carbon atom Chemical group C* 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 229920006395 saturated elastomer Polymers 0.000 claims description 6
- OTNVGWMVOULBFZ-UHFFFAOYSA-N sodium;hydrochloride Chemical compound [Na].Cl OTNVGWMVOULBFZ-UHFFFAOYSA-N 0.000 claims description 6
- 229910052717 sulfur Inorganic materials 0.000 claims description 6
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 6
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 4
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims description 4
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 4
- 229910052794 bromium Inorganic materials 0.000 claims description 4
- 239000000460 chlorine Substances 0.000 claims description 4
- 229910052801 chlorine Inorganic materials 0.000 claims description 4
- LMPDLIQFRXLCMO-UHFFFAOYSA-L dipotassium;hydrogen phosphate;phosphoric acid Chemical compound [K+].[K+].OP(O)(O)=O.OP([O-])([O-])=O LMPDLIQFRXLCMO-UHFFFAOYSA-L 0.000 claims description 4
- 229910052731 fluorine Inorganic materials 0.000 claims description 4
- 239000011737 fluorine Substances 0.000 claims description 4
- 229910052736 halogen Inorganic materials 0.000 claims description 4
- XMBWDFGMSWQBCA-UHFFFAOYSA-M iodide Chemical compound [I-] XMBWDFGMSWQBCA-UHFFFAOYSA-M 0.000 claims description 4
- 229910021645 metal ion Inorganic materials 0.000 claims description 4
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 4
- 229910001414 potassium ion Inorganic materials 0.000 claims description 4
- 229910052711 selenium Inorganic materials 0.000 claims description 4
- 229910001415 sodium ion Inorganic materials 0.000 claims description 4
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 claims description 4
- 125000004434 sulfur atom Chemical group 0.000 claims description 4
- 125000003944 tolyl group Chemical group 0.000 claims description 4
- ALVOEUTXKIPYGD-UHFFFAOYSA-M potassium;3-carboxy-3,5-dihydroxy-5-oxopentanoate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [K+].OC(=O)CC(O)(C(O)=O)CC(O)=O.OC(=O)CC(O)(C(O)=O)CC([O-])=O ALVOEUTXKIPYGD-UHFFFAOYSA-M 0.000 claims description 3
- WVULZDFWPQCPPJ-UHFFFAOYSA-N potassium;hydrochloride Chemical compound Cl.[K] WVULZDFWPQCPPJ-UHFFFAOYSA-N 0.000 claims description 3
- 229910052714 tellurium Inorganic materials 0.000 claims description 3
- SPRBJFWIUVWXBT-UHFFFAOYSA-K tripotassium 2-hydroxypropane-1,2,3-tricarboxylate 2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [K+].[K+].[K+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O SPRBJFWIUVWXBT-UHFFFAOYSA-K 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims 2
- 239000011591 potassium Substances 0.000 claims 2
- 229960003975 potassium Drugs 0.000 claims 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 1
- 229910052700 potassium Inorganic materials 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
- 239000007853 buffer solution Substances 0.000 abstract description 7
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 4
- 238000013399 early diagnosis Methods 0.000 description 4
- 239000011669 selenium Substances 0.000 description 4
- 239000011593 sulfur Substances 0.000 description 4
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 238000001839 endoscopy Methods 0.000 description 2
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- 229910052760 oxygen Inorganic materials 0.000 description 2
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- 238000002360 preparation method Methods 0.000 description 2
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- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 2
- 0 CC[*@@]1c(cccc2)c2NC1=CC(C)=Cc1*(CC)c(cccc2)c2[n]1 Chemical compound CC[*@@]1c(cccc2)c2NC1=CC(C)=Cc1*(CC)c(cccc2)c2[n]1 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
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- 238000000149 argon plasma sintering Methods 0.000 description 1
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- 238000013276 bronchoscopy Methods 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000002594 fluoroscopy Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
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- 229910052740 iodine Inorganic materials 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium atom Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Definitions
- the invention belongs to the field of medicine, and in particular relates to a supramolecular probe which can be used for diagnosing cancer. Background technique
- Sampling methods such as serum and stool detection are slightly more complicated, the accuracy is not good enough, and there is a certain false positive rate; for breast cancer, there are visual examination, palpation, mammography, B-ultrasound, breast light scattering imaging, Breast duct endoscopy, breast MRI and pathological biopsy, etc., but these methods require good operational skills and rich clinical experience, is a more subjective judgment method, can not produce a quantitative result, which increases The cost of detection and the difficulty of detection.
- Cyanine dye is a commonly used dye with a unique light-sensitive quality that has been unique for centuries. Physical and chemical properties have been discovered and are increasingly used as developers, photo-imaging agents, nonlinear optical materials, and the like.
- the inventors have designed a class of cyanine dyes which can specifically bind to diseased tissues of various cancers through extensive research, and are used for detection of various cancers. Summary of the invention
- One aspect of the invention provides a method of diagnosing cancer, the method comprising the steps of:
- step (1) adding a suitable amount of the compound of formula I to the single cell suspension obtained in step (1) such that the concentration of the compound of formula I in the single cell suspension is from 0.5 ⁇ to 200 ⁇ , and the obtained single cell suspension is first Incubate at 25-40 ° C for 10 minutes to 8 hours, then incubate at 2-8 ° C for 10 minutes to 8 hours;
- R 6 is alkyl, phenyl, alkyl-substituted phenyl
- R 2, R 3, and R 5 are independently selected from H or dC 6 alkyl, or R 2 and R 3 are attached to them
- the carbon atoms together form a 5- to 7-membered ring structure, or form a 5- to 7-membered ring structure together with R 5 and the carbon atom to which they are attached
- R 7 is a dC 6 alkyl or sulfonate substituted dC 6 alkyl
- Y is a counter ion, and varies according to the charge of R 7 .
- R 7 is an alkyl group
- Y is a halogen anion; if only one of R 7 has a sulfonate, Y is not required.
- a counterion if both R 7 and a sulfonate, Y is a triethylamine cation;
- Xi, X 2 is independently selected from carbon (C), oxygen (0), sulfur (S), selenium (Se) or ⁇ (Te).
- the buffer solution is a buffer containing monovalent metal ions, preferably a buffer containing potassium ions or sodium ions, including but not limited to sodium phosphate-sodium hydrogen phosphate buffer, potassium phosphate-hydrogen phosphate Potassium buffer, barbital sodium-hydrochloric acid buffer, barbital potassium-hydrochloric acid buffer, citric acid-sodium citrate buffer or citric acid-potassium citrate buffer.
- a buffer containing monovalent metal ions preferably a buffer containing potassium ions or sodium ions, including but not limited to sodium phosphate-sodium hydrogen phosphate buffer, potassium phosphate-hydrogen phosphate Potassium buffer, barbital sodium-hydrochloric acid buffer, barbital potassium-hydrochloric acid buffer, citric acid-sodium citrate buffer or citric acid-potassium citrate buffer.
- alkyl group of dC 6 is a linear or branched alkyl group having 1 to 6 carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, and Butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl.
- the process according to the invention which is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl, isohexyl, phenyl , methylphenyl or dimethylphenyl.
- R 2 , R 3 , R 4 and R 5 are independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, Isoamyl, n-hexyl or isohexyl.
- R 2 and R 3 and the carbon atom to which they are attached may form a 5- to 7-membered saturated ring structure or an unsaturated ring structure, which may or may not contain nitrogen (N) or Sulfur (S) atom.
- R 5 and the carbon atom to which they are attached may form a 5- to 7-membered saturated or unsaturated ring structure, which may or may not contain N or S atoms.
- Y is preferably a fluorine, chlorine, bromine, iodine anion or triethylamine cation.
- the method of the present invention enables early diagnosis of cancers such as lung cancer, colon cancer, and breast cancer.
- the staining rate of the lesion tissue cell sample when the staining rate of the lesion tissue cell sample is greater than 20%, preferably greater than 40%, more preferably greater than 60%, further preferably greater than 80%, the subject may be considered to have lung cancer;
- the staining rate of the colon cells of the subject when the staining rate of the colon cells of the subject is greater than 40%, preferably greater than 60%, more preferably greater than 80%, the subject may be considered to have colon cancer; in the case of diagnosis of breast cancer, The staining rate of the subject's mammary gland cells When the staining rate is greater than 25%, preferably greater than 40%, more preferably greater than 60%, the subject may be considered to have breast cancer.
- kits and systems for practicing the methods of the invention comprising the kit and a flow cytometer.
- kit and system comprising reagent I and reagent II, wherein reagent I is a buffer having a pH of 6-8, and reagent II is a compound of formula I below.
- R 6 is alkyl, phenyl, alkyl-substituted phenyl
- R 2, R 3, and R 5 are independently selected from H or dC 6 alkyl, or R 2 and R 3 are attached to them
- the carbon atoms together form a 5- to 7-membered ring structure, or form a 5- to 7-membered ring structure together with R 5 and the carbon atom to which they are attached
- R 7 is a dC 6 alkyl or sulfonate substituted dC 6 alkyl
- Y is a counter ion, and varies according to the charge of R 7 .
- R 7 is an alkyl group
- Y is a halogen anion; if only one of R 7 has a sulfonate, Y is not required.
- a counterion if both R 7 and sulfonate have a sulfonate, Y is a triethylamine cation; Xi,
- X 2 is independently selected from carbon (C), oxygen (0), sulfur (S), selenium (Se) or tellurium (Te).
- Kits and systems according to the present invention wherein for the compound of formula I, the preparation method can be referred to
- the buffer solution is a buffer containing monovalent metal ions, preferably a buffer containing potassium ions or sodium ions, including but not limited to sodium phosphate-sodium hydrogen phosphate buffer, potassium phosphate - Potassium hydrogen phosphate buffer, barbital sodium-hydrochloric acid buffer, barbital potassium-hydrochloric acid buffer, citric acid-sodium citrate buffer or citric acid-potassium citrate buffer.
- a buffer containing monovalent metal ions preferably a buffer containing potassium ions or sodium ions, including but not limited to sodium phosphate-sodium hydrogen phosphate buffer, potassium phosphate - Potassium hydrogen phosphate buffer, barbital sodium-hydrochloric acid buffer, barbital potassium-hydrochloric acid buffer, citric acid-sodium citrate buffer or citric acid-potassium citrate buffer.
- alkyl group of dC 6 is a linear or branched alkyl group having 1 to 6 carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, isopropyl Base, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl.
- Kit and system according to the present invention which are selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl, isohexyl , phenyl, methylphenyl or dimethylphenyl.
- Kits and systems according to the invention wherein R 2 , R 3 , R 4 and R 5 are independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, Pentyl, isopentyl, n-hexyl or isohexyl.
- Kits and systems according to the present invention wherein R 2 and R 3 and the carbon atom to which they are attached may form a 5- to 7-membered saturated or unsaturated ring structure, which may or may not contain nitrogen ( N) or sulfur (S) atom.
- Kits and systems according to the present invention wherein R 5 and the carbon atom to which they are attached may form a 5- to 7-membered saturated or unsaturated ring structure, which may or may not contain N or S atoms.
- Kits and systems according to the invention wherein Y is preferably a fluorine, chlorine, bromine, iodine anion or triethylamine cation.
- kits and systems of the invention can be used for the early diagnosis of cancers such as lung cancer, colon cancer, breast cancer and the like.
- the invention provides the use of a compound of formula I, in a kit and system for the early diagnosis of a cancer, such as lung cancer, colon cancer, breast cancer, etc., wherein the compound of formula I is as defined above.
- the methods, kits and systems provided by the present invention for diagnosing cancer have the advantages of: high sensitivity, innovative use of supramolecular fluorescent probes, sensitivity is more than loo times of conventional single molecule probes ;
- FIG. 1 is a flow chart showing the flow detection dyeing rate of a test sample obtained according to Example 1 of the present invention
- FIG. 2 is a flow chart showing the dyeing rate of a test sample obtained according to Example 6 of the present invention.
- Figure 3 is a flow chart showing the flow detection dyeing rate of the test sample obtained according to Example 9 of the present invention; and
- Figure 4 is a flow chart showing the dyeing rate of the test sample obtained according to Example 14 of the present invention.
- Figure 5 is a flow chart showing the flow detection dyeing rate of the test sample obtained according to Example 17 of the present invention
- Figure 6 is a flow chart showing the dyeing rate of the test sample obtained according to Example 22 of the present invention. summary graph.
- nude mice 50 nude mice were housed, 20 were used as blank controls, and 30 lung cancer cells (A549) were inoculated into nude mice. After the tumors were grown, the nude mice inoculated with A549 were taken for tumor tissue, and the blank control group was directly taken from lung cells.
- Single The cell suspension was washed and suspended with a sodium phosphate-sodium hydrogen phosphate buffer of pH 6, and 20 samples of normal cell suspension and 30 samples of tumor cell suspension were obtained, and the concentration of each cell suspension sample was 10 4 to 10 9 The range of cells/mL.
- Each sample was incubated for 1 hour in a 37 ° C incubator and then incubated for 1 hour in a 4 ° C freezer. After the incubation, the sample was centrifuged, the dye solution was removed, the cells were washed with a sodium phosphate-sodium hydrogen phosphate buffer of pH 6, and a single cell suspension of 10 3 to 10 6 cells/ml was prepared.
- Flow cytometry was used to detect the fluorescence signal of each sample at 564-606 nm, and the ratio of staining positive cells to the total number of cells, that is, the staining rate, was obtained.
- the staining rate of 50 cell samples obtained by the above method was statistically analyzed, and the results are shown in Fig. 1. Referring to Fig. 1, it can be clearly seen that the staining rate of all lung cancer samples is greater than 70%, and the staining rate of all normal lung cell samples is less than 10%. Therefore, normal lung cells and lungs can be easily used by the diagnostic method of the present embodiment. Tumor cells are distinguished.
- Tumor cells and normal lung cells of the above 50 nude mice were examined by the same procedure as in Example 1, except that the compounds used were:
- the final concentration of the compound in each sample was 50 ⁇ , the incubation temperature and time were 15 minutes at 37 ° C, and 15 minutes at 4 V.
- Flow cytometry was used to detect the fluorescence signal of each sample above 670 nm, thereby obtaining the staining rate of each sample. , the dyeing rate of each sample was obtained.
- Test results the staining rate of all lung cancer samples was greater than 75%, and the staining rate of all normal lung cell samples was less than 15%. Therefore, the normal lung cells and lung tumor cells can be easily distinguished by the diagnosis method of the present embodiment.
- Example 2 Using the same procedure as in Example 1, the tumor cells and normal lung cells of the above 50 nude mice were examined, and the difference was made.
- the final concentration of the compound in each sample was: 150 ⁇ , the incubation temperature and time were 8 hours at 37 ° C, and 1 hour at 4 V. Fluorescence signals of 670 nm or more of each sample were detected by flow cytometry to obtain the staining rate of each sample. .
- test results the staining rate of all lung cancer samples is greater than 85%, and the staining rate of all normal lung cell samples is less than 20%. Therefore, the normal lung cells and lung tumor cells can be easily distinguished by the diagnostic method of the present embodiment. Open.
- Tumor cells and normal lung cells of the above 50 nude mice were examined by the same procedure as in Example 1, and the difference was as follows:
- the final concentration of the compound in each sample was: 80 ⁇ , the incubation temperature and time were 2 hours at 37 ° C, 7.5 hours at 4 ° C, and the fluorescence signal in the range of 564-606 nm was detected by flow cytometry. The staining rate of each sample.
- test results the staining rate of all lung cancer samples was greater than 80%, and the staining rate of all normal lung cell samples was less than 20%. Therefore, the normal lung cells and lung tumor cells can be easily distinguished by the diagnosis method of the present embodiment.
- Tumor cells and normal lung cells of the above 50 nude mice were examined by the same procedure as in Example 1, except that the compounds used were:
- the final concentration of the compound in each sample was 0.5 ⁇ , the incubation temperature and time were 4 hours at 37 ° C, and 4 hours at 4 ° C. Fluorescence signals of 670 nm or more of each sample were detected by flow cytometry to obtain each sample. Staining rate.
- HEF human lung fibroblasts
- RAW264.7 murine alveolar macrophages
- HaCaT human skin cells
- IH3T3 mouse embryonic fibroblasts
- CHO hamster ovary cells
- the tumor cell line and the normal cell line are respectively made into a single cell suspension, washed and suspended with a pH 8 citric acid-sodium citrate buffer solution, and a normal cell suspension sample and a tumor cell suspension sample are obtained, and the concentration of each cell suspension sample is obtained. In the range of 10 4 ⁇ 10 9 cells / mL.
- Each sample was incubated for 0.5 hour in a 37 ° C incubator and then incubated for an additional 0.5 hours in a 4 ° C freezer.
- the sample was centrifuged, the dye solution was removed, and the cells were washed with a pH 8 citric acid-sodium citrate buffer and resuspended to obtain a single cell suspension of 10 3 to 10 6 cells/ml.
- Flow cytometry was used to detect the fluorescence signal of each sample at 564-606 nm, and the ratio of staining positive cells to the total number of cells, that is, the staining rate, was obtained.
- the staining rate of all lung cancer cell samples is greater than 66%, and all normal lung cell samples are less than 25%. Therefore, normal lung cells and lung tumor cells can be easily used by the diagnostic method of the present embodiment. differentiate.
- the lung cancer cells and normal cell lines were tested in the same manner as in Example 6, except that the use was performed.
- the compound had a final concentration of 50 ⁇ M in each sample, and the incubation temperature and time were 37 ° C for 10 minutes.
- the fluorescence signal of the sample above 670 nm was detected by flow cytometry to obtain the staining rate of each sample.
- the final concentration of the compound in each sample was 200 ⁇ , the incubation temperature and time were 0.5 hours at 37 ° C, and 0.5 hour at 4 ° C.
- the fluorescence signal of the sample above 564-606 nm was detected by flow cytometry to obtain each sample. Staining rate.
- the staining rate of all lung cancer cell samples was greater than 70%, and all normal lung cell samples were less than 25%. Therefore, the normal lung cells and lung tumor cells can be easily distinguished by the diagnostic method of the present embodiment.
- mice 50 nude mice were housed, 20 were used as blank controls, and 30 colon cancer cells (GEO) were inoculated into nude mice. After the tumors were grown, the nude mice inoculated with GEO took tumor tissue, and the blank control group directly took colon tissue cells.
- the single cell suspension was prepared, washed and suspended with a sodium phosphate-sodium hydrogen phosphate buffer solution of pH 6, and 20 samples of normal cell suspension and 30 samples of tumor cell suspension were obtained, and the concentration of each cell suspension sample was 10 4 ⁇ A range of 10 9 cells/mL.
- the compound of the formula was added to each sample so that the final concentration of the compound in each sample was 6 ⁇ .
- Each sample was incubated for 0.5 hour in a 37 ° C incubator and then incubated for an additional 0.5 hours in a 4 ° C refrigerator. After the incubation, the sample was centrifuged, the dye solution was removed, the cells were washed with a sodium phosphate-sodium hydrogen phosphate buffer of pH 6, and a single cell suspension of 10 3 to 10 6 cells/ml was prepared.
- Flow cytometry was used to detect the fluorescence signal of each sample at 564-606 nm, and the ratio of staining positive cells to the total number of cells, that is, the staining rate, was obtained.
- the staining rate of 50 cell samples obtained by the above method was statistically analyzed, and the results are shown in Fig. 3. Referring to Fig. 3, it can be clearly seen that the staining rate of all colon cancer samples is greater than 85%, and the staining rate of all control samples is less than 40%. Therefore, normal cell and colon cancer can be easily induced by the diagnostic method of the present embodiment.
- the tumor cells are separated.
- Tumor cells and normal cells of the above 50 nude mice were examined by the same procedure as in Example 9, except that the compounds used were:
- the final concentration of the compound in each sample was 2 ⁇ , the incubation temperature and time were 4 hours at 37 ° C, and 4 hours at 4 ° C. Fluorescence signals of 670 nm or more of each sample were detected by flow cytometry to obtain staining of each sample. rate.
- the staining rate of all colon cancer samples was more than 75%, and the staining rate of all the control samples was less than 35%. Therefore, the normal cells and colon cancer-induced tumor cells can be easily separated by the diagnosis method of the present embodiment.
- the final concentration of the compound in each sample was: 50 ⁇ M, the incubation temperature and time were 37 ° C for 10 minutes, and the fluorescence signal of each sample was detected by flow cytometry at 670 nm or more to obtain the staining rate of each sample.
- Tumor cells and normal cells of the above 50 nude mice were examined in the same manner as in Example 9.
- the staining rate of all colon cancer samples was more than 55%, and the staining rate of all the control samples was less than 20%. Therefore, the normal cells and colon cancer-induced tumor cells can be easily separated by the diagnosis method of the present embodiment.
- Tumor cells and normal cells of the above 50 nude mice were examined by the same procedure as in Example 9, and the difference was as follows:
- the staining rate of all colon cancer samples was more than 85%, and the staining rate of all the control samples was less than 40%. Therefore, the normal cells and colon cancer-induced tumor cells can be easily separated by the diagnosis method of the present embodiment.
- Colon cancer cells are cultured.
- CHO hamster ovary cells
- NASH mouse embryonic fibroblasts
- HVF human lung fibroblasts
- HUMC human vascular smooth muscle cells
- HaCaT human skin cells
- the normal cell line is made into a single cell suspension, washed and suspended with a pH 8 citrate-potassium citrate buffer solution to obtain a normal cell suspension sample and a tumor cell suspension sample, and the concentration of each cell suspension sample is ⁇ 4 ⁇ ⁇ 9 cells
- each sample was incubated for 1 hour in a 37 ° C incubator and then incubated for 1 hour in a 4 ° C freezer. After the incubation, each sample was centrifuged, the dye solution was removed, and the cells were washed with a pH 8 citrate-potassium citrate buffer solution and resuspended to obtain a single cell suspension of 10 3 to 10 6 cells/ml.
- Flow cytometry was used to detect the fluorescence signal of each single cell suspension at 564-606 nm, and the ratio of staining positive cells to the total number of cells in each sample, that is, the staining rate, was obtained.
- the staining rate of all colon cancer cell samples is greater than 40%, and the staining rate of all normal lung cell samples is less than 25%. Therefore, normal lung cells can be easily used by the diagnostic method of the present embodiment. Separated from lung tumor cells.
- the final concentration of the compound in each sample was 0.5 ⁇ , the incubation temperature and time were 4 hours at 37 ° C, and 4 hours at 4 ° C. Fluorescence signals of 670 nm or more of each sample were detected by flow cytometry to obtain each sample. Staining rate.
- the staining rate of all colon cancer cell samples was more than 45%, and the staining rate of all normal lung cell samples was less than 25%. Therefore, the normal lung cells and lung tumor cells can be easily separated by the diagnostic method of this embodiment.
- the final concentration of the compound in each sample was 200 ⁇ , the incubation temperature and time were 15 minutes at 37 ° C, and 15 minutes at 4 ° C.
- the fluorescence signal of each sample in the range of 564-606 nm was detected by flow cytometry. The staining rate of the sample.
- the staining rate of all colon cancer cell samples was more than 90%, and the staining rate of all normal lung cell samples was less than 35%. Therefore, the normal lung cells and lung tumor cells can be easily separated by the diagnostic method of the present embodiment.
- MCF-7 breast cancer cells
- Each sample was incubated for 1 hour in a 37 ° C incubator and then incubated for 1 hour in a 4 ° C freezer. After the incubation, the sample was centrifuged, the dye solution was removed, the cells were washed with a sodium phosphate-sodium hydrogen phosphate buffer of pH 6, and resuspended to obtain a single cell suspension of 10 3 to 10 6 cells/ml.
- the fluorescence signal of the cells at 564-606 nm was measured by flow cytometry to obtain the staining rate of each sample.
- the staining rate of 50 cell samples obtained by the above method was statistically analyzed, and the results are shown in Fig. 5. Referring to Fig. 5, it can be clearly seen that the staining rate of all breast cancer samples is greater than 95%, and the staining rate of all control samples is less than 25%, so that normal cells and breast cancer can be easily used by the diagnostic method of the present embodiment.
- the induced tumor cells are separated.
- Tumor cells and normal cells of the above 50 nude mice were examined by the same procedure as in Example 17, except that the compounds used were:
- the concentration of the compound in the sample was 50 ⁇ M, the incubation temperature and time were 15 minutes at 37 ° C, and 15 minutes at 4 ° C, and then the fluorescence signal of the sample was measured by flow cytometry at a wavelength of 670 nm or more to obtain the staining rate of each sample.
- Test results All breast cancer samples were stained at more than 90%, and all control samples were stained at less than 20%.
- Example 17 The same procedure as in Example 17 was used to detect the tumor cells and normal cells of the above 50 nude mice, and the difference was made.
- the concentration of the compound was: 10 ⁇ , the incubation temperature and time were 7.5 hours at 37 ° C, and 0.5 hour at 4 ° C, and the fluorescence signal above 670 nm was detected by flow, and the staining rate of each sample was obtained.
- Test results All breast cancer samples were stained at more than 80%, and all control samples were stained at less than 25%.
- Tumor cells and normal cells of the above 50 nude mice were examined by the same procedure as in Example 17, except that the compounds used were:
- the concentration of the compound was: 6 ⁇ M, the incubation temperature and time were 10 minutes at 37 ° C, and 8 hours at 4 ° C, and the fluorescence signal in the range of 564-606 nm was detected by flow cytometry.
- Test results All breast cancer samples were stained at more than 70%, and all control samples were stained at less than 25%.
- Example 17 The same procedure as in Example 17 was used to detect the tumor cells and normal cells of the above 50 nude mice, and the difference was made.
- the final concentration of the compound in each sample was 2 ⁇ M, the incubation temperature and time were 1 hour at 37 ° C, and 1 hour at 4 ° C. Fluorescence signals of 670 nm or more of each sample were detected by flow cytometry. Test results: The staining rate of all breast cancer samples was above 80%, and the staining rate of all control samples was below 25%.
- CHO hamster ovary cells
- NASH mouse embryonic fibroblasts
- HVF human lung fibroblasts
- HUMC human vascular smooth muscle cells
- HaCaT human skin cells
- Each sample was incubated for 4 hours in a 37 ° C incubator and then incubated for 4 hours in a 4 ° C freezer. After the incubation, the sample was centrifuged, the dye solution was removed, and the cells were washed with a barbital sodium-hydrochloric acid buffer of pH 8, to prepare a single cell suspension of 10 3 to 10 6 cells/ml.
- the fluorescence signal of the cells at 564-606 nm was measured by flow cytometry to obtain a ratio of staining positive cell clusters, i.e., staining rate.
- the staining rate of all breast cancer cell samples is greater than 40%, and the staining rate of all normal breast cell samples is less than 25%. Therefore, normal breast cells can be easily used by the diagnostic method of the present embodiment. It is distinguished from breast cancer cells.
- the final concentration of the compound in each sample was 200 ⁇ , the incubation temperature and time were 7.5 hours at 37 ° C, and 0.5 hour at 4 ° C. Fluorescence signals of 670 nm or more of each sample were detected by flow cytometry to obtain staining of each sample. rate.
- Test results The staining rate of all breast cancer cell samples was greater than 45%, and the staining rate of all normal breast cell samples was less than 25%.
- Example 22 The same procedure as in Example 22 was carried out to detect breast cancer cells and normal cell lines, with the difference that
- the final concentration of the compound in each sample was 1 ⁇ , and the incubation temperature and time were 2 hours at 37 ° C, 4 ° C
- the fluorescence signal at 564-606 nm of each sample was detected by flow cytometry to obtain the staining rate of each sample.
- Test results The staining rate of all breast cancer cell samples was greater than 35%, and the staining rate of all normal breast cell samples was less than 20%.
- the flow cytometer used in the embodiment of the present invention is a FACSCalibur model flow cytometer manufactured by Becton, Dickinson and Company CBD, which has fluorescence excitation wavelengths of 488 nm and 688 nm, but convection in the present invention.
- the type of the cytometer is not limited as long as the method of the present invention can be carried out; the concentration of each cell suspension sample is not particularly limited in the method of the present invention, as long as the flow cytometer can be satisfied.
- the detection requirements are sufficient, and those skilled in the art can determine the concentration of the cell suspension as needed.
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Abstract
本发明提供一种诊断癌症的方法，所述方法包括以下步骤：（1）取得受试者的肺部病灶组织样品，并用pH值为6~8的缓冲液将取得的组织样品制备成浓度为104~109个细胞/mL的单细胞悬液；（2）向步骤（1）中获得的单细胞悬液中加入式I的化合物以使单细胞悬液中式I的化合物浓度为0.5μΜ至200μΜ，并将获得的单细胞悬液先在25-40°C下孵育10分钟至8小时，然后在2-8°C下孵育10分钟至8小时；（3）收集孵育后的细胞，用pH值为6~8的缓冲液洗涤，并重新悬浮，得到浓度为103~106个细胞/ml的单细胞悬液；（4）用流式细胞仪在515nm~650nm或670nm以上波长范围内对步骤（3）中获得的细胞悬液的荧光信号进行检测，计算细胞的染色率，所述染色率为有荧光信号产生的细胞占细胞总数的比例；当染色率大于20%，优选大于40%，更优选大于60%，进一步优选大于80%时，则可认定受试者患有癌症。
Description
诊断癌症的方法、 试剂盒和系统
相关申请的交叉引用
本申请要求 2012年 5月 24日提交的申请号为 201210164349.X和 201210164341.3 以及 201210164367.8的中国专利申请的优先权。 本申请通过引用包括上述申请的全 部内容。 技术领域
本发明属于医药领域， 具体而言涉及一种可用于诊断癌症的超分子探针。 背景技术
今年来随着人们生活节奏的加快、环境的恶化， 癌症逐渐成为影响人类健康和寿 命的主要杀手。 例如肺癌、 结肠癌、 乳腺癌等每年造成数百万人死亡。 根据癌症的种 类不同， 其诊断方法也有所不同。
在临床中癌症的诊断方法有很多， 例如针对肺癌有 X线检查、 支气管镜检、 放 射性核素检查、 痰液细胞学检查、 剖胸探查术、 ECT 检查及纵膈镜检查等等， 但这 些方法绝大多数都是在肺癌晚期才有比较好的检测效果，若用于早期则灵敏度和准确 度不高。并且这些现有的临床方法都需要丰富的临床经验和操作技巧，增加了检测成 本和检测难度； 针对结肠癌主要的诊断方法有 X线检查， 内镜检查， 血清癌胚抗原 (CEA)检查， B型超声扫描， 粪便检查等。 但这些方法多数都需要较好的操作技巧以 及丰富的临床经验， 是比较主观的判断方法。取样简单的方法如血清和粪便检测操作 略复杂， 准确率不够好， 且有一定的假阳性率； 针对乳腺癌有视诊、 触诊、 乳腺 X 线检查、 B超检查、 乳腺光散射成像、 乳管内视镜、 乳腺 MRI检查以及病理学活检 等等，但这些方法都需要较好的操作技巧以及丰富的临床经验， 是一种比较主观的判 断方法， 无法产生一个量化的结果， 这就增加了检测成本和检测难度。
并且， 癌症的早期症状通常不明显， 非常容易出现误诊， 所以当患者确诊时通常 已进入癌症晚期。 因此， 研究开发新的癌症诊断尤其是早期诊断是非常必要的。
菁染料是一种常用染料， 具有独特的光敏感性质， 几个世纪以来， 伴随着其独特
理化、 光学性质被发现， 逐渐被用作显影剂， 光影剂， 非线性光学材料等。
本发明人通过大量的研究，设计了一类可与多种癌症的病变组织能够特异性地结 合的菁染料， 用于多种癌症的检测。 发明内容
本发明的一个方面提供一种诊断癌症的方法， 所述方法包括以下步骤：
( 1 ) 取得受试者的病灶组织样品， 并用 pH值为 6~8的缓冲液将取得的组织样 品制备成浓度为 104~109个细胞 /mL的单细胞悬液；
(2) 向步骤（1 ) 中获得的单细胞悬液中加入适量的式 I的化合物以使单细胞悬 液中式 I的化合物浓度为 0.5μΜ至 200μΜ， 并将获得的单细胞悬液先在 25-40°C下孵 育 10分 至 8小时， 然后在 2-8°C下孵育 10分钟至 8小时；
(3 ) 收集孵育后的细胞， 用 pH值为 6~8的缓冲液洗涤， 并重新悬浮， 得到浓 度为 103~106个细胞 /ml的单细胞悬液；
(4)用流式细胞仪在 515nm~650nm或 670nm以上波长范围内对步骤（3 ) 中获 得的细胞悬液的荧光信号进行检测， 计算有荧光信号产生的细胞占细胞总数的比例， 即细胞的染色率； 当染色率大于 20%， 优选大于 40%， 更优选大于 60%， 进一步优 选大于 80%时， 则可认定受试者患有癌症；
其中： 《 为 6的烷基、 苯基、 烷基取代的苯基； R2、 R3、 和 R5独立地选 自 H或 d-C6的烷基， 或者 R2和 R3与它们所连接的碳原子一起形成 5元至 7元的环 结构， 或者 和 R5与它们所连接的碳原子一起形成 5元至 7元的环结构； 和 R7 为 d-C6烷基或者磺酸基取代的 d-C6烷基； Y为反离子， 根据 和 R7所带电荷的 不同而不同， 若 和 R7为烷基， 则 Y为卤素阴离子； 若 和 R7只有一个带有磺酸 根， 则无需 Y作为反离子； 若 和 R7均带有磺酸根， 则 Y为三乙胺阳离子； Xi， X2独立地选自碳 （C)、 氧 （0)、 硫 （S)、 硒 （Se) 或碲 （Te)。
根据本发明的方法其中对于式 I的化合物而言，其制备方法可以参考 Leslie G S., Brooker and Frank L. W., JACS, 1935, 547-551中记载的合成路线， 也可以使用本领域 熟知的其他方法来制备。
根据本发明的方法， 其中所述缓冲溶液为含有一价金属离子的缓冲液， 优选含有 钾离子或钠离子的缓冲液， 包括但不限于磷酸钠 -磷酸氢钠缓冲液、 磷酸钾-磷酸氢钾 缓冲液、 巴比妥钠-盐酸缓冲液、 巴比妥钾-盐酸缓冲液、柠檬酸-柠檬酸钠缓冲液或柠 檬酸 -柠檬酸钾缓冲液。
根据本发明的方法， 其中 d-C6的烷基为碳原子数为 1-6的直链或支链的烷基， 包括但不限于， 甲基、 乙基、 正丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异 戊基、 正己基或异己基等。
根据本发明的方法， 其中 选自甲基、 乙基、 正丙基、 异丙基、 正丁基、 异丁 基、 叔丁基、 戊基、 异戊基、 正己基、 异己基、 苯基、 甲基苯基或二甲基苯基。
根据本发明的方法， 其中 R2、 R3、 R4和 R5独立地选自甲基、 乙基、 正丙基、 异 丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基或异己基。
根据本发明的方法， 其中 R2和 R3与它们所连接的碳原子可以形成 5元至 7元的 饱和环结构或不饱和环结构， 所述环结构可以含或不含有氮 （N) 或硫 （S) 原子。
根据本发明的方法， 其中 和 R5与它们所连接的碳原子可以形成 5元至 7元的 饱和或不饱和环结构， 所述环结构可以含或不含有 N或 S原子。
根据本发明的方法， 其中 Y优选为氟、 氯、 溴、 碘阴离子或三乙胺阳离子。 本发明的方法可以对肺癌、 结肠癌、 乳腺癌等癌症进行早期诊断。
在诊断肺癌的情况下， 当病灶组织细胞样品的染色率大于 20%， 优选大于 40%， 更优选大于 60%， 进一步优选大于 80%时， 则可认定受试者患有肺癌； 在诊断结肠 癌的情况下， 当受试者的结肠细胞的染色率大于 40%， 优选大于 60%， 更优选大于 80%时， 可认定受试者患有结肠癌； 在诊断乳腺癌的情况下， 当受试者的乳腺细胞的 染色率当染色率大于 25%时， 优选大于 40%时， 更优选大于 60%时， 可认定受试者 患有乳腺癌。
本发明的另一个方面是提供用于实施本发明方法的试剂盒和系统，所述系统包括 所述试剂盒和流式细胞仪。
根据本发明的试剂盒和系统， 其中所述试剂盒包括试剂 I和试剂 II， 其中试剂 I 为 pH值为 6~8的缓冲液， 试剂 II为下式 I所示的化合物
式 I
其中： 《 为 6的烷基、 苯基、 烷基取代的苯基； R2、 R3、 和 R5独立地选 自 H或 d-C6的烷基， 或者 R2和 R3与它们所连接的碳原子一起形成 5元至 7元的环 结构， 或者 和 R5与它们所连接的碳原子一起形成 5元至 7元的环结构； 和 R7 为 d-C6烷基或者磺酸基取代的 d-C6烷基； Y为反离子， 根据 和 R7所带电荷的 不同而不同， 若 和 R7为烷基， 则 Y为卤素阴离子； 若 和 R7只有一个带有磺酸 根， 则无需 Y作为反离子； 若 和 R7均带有磺酸根， 则 Y为三乙胺阳离子； Xi，
X2独立地选自碳 （C)、 氧 （0)、 硫 （S)、 硒 （Se) 或碲 （Te)。
根据本发明的试剂盒和系统， 其中对于式 I的化合物而言， 其制备方法可以参考
Leslie G. S., Brooker and Frank L. W.， JACS, 1935, 547-551中记载的合成路线， 也可以 使用本领域熟知的其他方法来制备。
根据本发明的试剂盒和系统， 其中所述缓冲溶液为含有一价金属离子的缓冲液， 优选含有钾离子或钠离子的缓冲液， 包括但不限于磷酸钠 -磷酸氢钠缓冲液、 磷酸钾- 磷酸氢钾缓冲液、 巴比妥钠-盐酸缓冲液、 巴比妥钾-盐酸缓冲液、柠檬酸 -柠檬酸钠缓 冲液或柠檬酸 -柠檬酸钾缓冲液。
根据本发明的试剂盒和系统， 其中 d-C6的烷基为碳原子数为 1-6的直链或支链 的烷基， 包括但不限于， 甲基、 乙基、 正丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基或异己基等。
根据本发明的试剂盒和系统， 其中 选自甲基、 乙基、 正丙基、 异丙基、 正丁 基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基、 异己基、 苯基、 甲基苯基或二甲基苯 基。
根据本发明的试剂盒和系统， 其中 R2、 R3、 R4和 R5独立地选自甲基、 乙基、 正 丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基或异己基。
根据本发明的试剂盒和系统， 其中 R2和 R3与它们所连接的碳原子可以形成 5元 至 7元的饱和环结构或不饱和环结构， 所述环结构可以含或不含有氮 （N) 或硫 （S)
原子。
根据本发明的试剂盒和系统， 其中 和 R5与它们所连接的碳原子可以形成 5元 至 7元的饱和或不饱和环结构， 所述环结构可以含或不含有 N或 S原子。
根据本发明的试剂盒和系统， 其中 Y优选为氟、 氯、 溴、 碘阴离子或三乙胺阳 离子。
本发明的试剂盒和系统可以用于肺癌、 结肠癌、 乳腺癌等癌症的早期诊断。 本发明的有一方面提供式 I的化合物在制备用于癌症如肺癌、 结肠癌、 乳腺癌等 的早期诊断的试剂盒和系统中的用途， 其中式 I的化合物如前文所定义。
与现有技术相比，本发明提供的用于诊断癌症的方法、试剂盒和系统的优点在于: 高灵敏度， 创新性的采用超分子荧光探针， 灵敏度是传统单分子探针的 loo倍以 上；
高特异性， 正常细胞和癌细胞染色率差别很大， 准确率约 97~99%。
仅需一步染色反应， 无需制作切片等， 操作简单， 检测时间短；
结果为量化数值， 无需描述， 无需经验， 更加客观准确。 附图说明 图 1是表示根据本发明实施例 1得到的受试样品的流式检测染色率统计图； 图 2是表示根据本发明实施例 6得到的受试样品的流式检测染色率统计图； 图 3是表示根据本发明实施例 9得到的受试样品的流式检测染色率统计图； 图 4是表示根据本发明实施例 14得到的受试样品的流式检测染色率统计图； 图 5是表示根据本发明实施例 17得到的受试样品的流式检测染色率统计图； 图 6是表示根据本发明实施例 22得到的受试样品的流式检测染色率统计图。 具体实施方式 下面将参照附图以具体实施例的方式来更详细地描述本发明， 但是应当理解， 本 发明可以以不同的方式实施，提供这些实施例仅是为了使本说明书充分和完整， 以使 本领域技术人员能够实施本发明， 本发明的范围不应当限定为本文所列的具体实施 例。
实施例 1
饲养 50只裸鼠， 20只作为空白对照， 30只接种肺癌细胞 （A549) 于裸鼠体内， 待肿瘤长成后， 接种 A549的裸鼠取肿瘤组织， 空白对照组直接取肺细胞， 制成单细
胞悬液， 用 pH6的磷酸钠-磷酸氢钠缓冲液洗涤、悬浮， 得到 20份正常细胞悬液样品 和 30份肿瘤细胞悬液样品， 各细胞悬液样品的浓度在 104~109个细胞 /mL的范围。
为 ΙΟμΜ
每份样品在 37°C培养箱中孵育 1小时， 然后在 4°C冰箱中继续孵育 1小时。 孵育结束后将样品离心， 去除染料溶液， 用 pH6的磷酸钠-磷酸氢钠缓冲液洗涤 细胞， 并制备 103~106个细胞 /ml的单细胞悬液。
用流式细胞仪检测各样品在 564-606nm处的荧光信号，获得染色阳性细胞占细胞 总数的比例， 即染色率。
对通过以上方式获得的 50份细胞样品的染色率进行统计分析，结果如图 1所示。 参照图 1， 可以清楚地看出所有肺癌样品染色率大于 70%， 所有正常肺细胞样品 的染色率均小于 10%， 因此，采用本实施例的诊断方式可以很容易地将正常肺细胞和 肺肿瘤细胞区分开。
实施例 2
采用与实施例 1相同的步骤对以上 50只裸鼠的肿瘤细胞和正常肺细胞进行检测， 区别在于， 所使用化合物为：
该化合物在各样品中的终浓度为 50μΜ， 孵育温度和时间为 37°C下 15分钟， 4V 下 15分钟， 用流式细胞仪检测各样品 670nm以上的荧光信号， 从而获得各样品的染 色率， 获得各样品的染色率。
检测结果：所有肺癌样品染色率大于 75%，所有正常肺细胞样品的染色率均小于 15%, 因此， 采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细胞区分 开。
实施例 3
采用与实施例 1相同的步骤对以上 50只裸鼠的肿瘤细胞和正常肺细胞进行检测， 区别
该化合物在各样品中的终浓度为： 150μΜ，孵育温度和时间为 37°C下 8小时， 4V 下 1小时， 用流式细胞仪检测各样品 670nm以上的荧光信号， 获得各样品的染色率。
检测结果：所有肺癌样品染色率大于 85%，所有正常肺细胞样品的染色率均小于 20%, 因此， 采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细胞区分
开。
实施例 4
采用与实施例 1相同的步骤对以上 50只裸鼠的肿瘤细胞和正常肺细胞进行检测， 区别 ：
检测结果：所有肺癌样品染色率大于 80%，所有正常肺细胞样品的染色率均小于 20%, 因此， 采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细胞区分 开。
实施例 5
采用与实施例 1相同的步骤对以上 50只裸鼠的肿瘤细胞和正常肺细胞进行检测， 区别在于， 所使用化合物为：
检测结果：所有肺癌样品染色率大于 45%，所有正常肺细胞样品的染色率均小于
15%, 因此， 采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细胞区分 开。
实施例 6
培养人肺癌细胞 (A549)。
培养正常细胞系， 包括人肺成纤维细胞 (HLF)， 鼠肺泡巨噬细胞 (RAW264.7), 人 皮肤细胞 (HaCaT), 鼠胚胎成纤维细胞 ( IH3T3), 仓鼠卵巢细胞 （CHO)， 将肿瘤细 胞株和正常细胞系分别制成单细胞悬液， 用 pH8的柠檬酸-柠檬酸钠缓冲液洗涤、 悬 浮， 得到正常细胞悬液样品和肿瘤细胞悬液样品， 各细胞悬液样品的浓度在 104~109 个细胞 /mL的范围。
向每份样品中加入终浓度为 ΙΟμΜ的试剂 II， 该化合物为
每份样品在 37°C培养箱中孵育 0.5小时， 然后在 4°C冰箱中继续孵育 0.5小时。
孵育结束后将样品离心， 去除染料溶液， 用 pH8的柠檬酸-柠檬酸钠缓冲液洗涤 细胞， 并重新悬浮， 获得 103~106个细胞 /ml的单细胞悬液。
用流式细胞仪检测各样品在 564-606nm处的荧光信号，获得染色阳性细胞占细胞 总数的比例， 即染色率。
对通过以上方式重复 30次获得的 30份体外培养细胞的染色率进行统计分析，结 果如图 2所示。
参照图 2， 可以清楚地看出所有肺癌细胞样品染色率大于 66%， 所有正常肺细胞 样品均小于 25%， 因此，采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿 瘤细胞区分开。
实施例 7
采用与实施例 6相同的步骤对肺癌细胞和正常细胞株进行检测， 区别在于， 所使 用化
检测结果:所有肺癌细胞样品染色率大于 50%，所有正常肺细胞样品均小于 20%，
因此， 采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细胞区分开。 实施例 8
采用与实施例 6相同的步骤对肺癌细胞和正常细胞株进行检测， 区别在于， 所使 用化 ：
该化合物在各样品中的终浓度为 200μΜ，孵育温度和时间为 37°C下 0.5小时， 4°C 下 0.5小时， 用流式细胞仪检测样品 564-606nm以上的荧光信号， 获得各样品的染色 率。
检测结果:所有肺癌细胞样品染色率大于 70%，所有正常肺细胞样品均小于 25%， 因此， 采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细胞区分开。
实施例 9
饲养 50只裸鼠， 20只作为空白对照， 30只接种结肠癌细胞（GEO)于裸鼠体内， 待肿瘤长成后， 接种 GEO的裸鼠取肿瘤组织， 空白对照组直接取结肠组织细胞， 制 成单细胞悬液， 用 pH6的磷酸钠-磷酸氢钠缓冲液洗涤、悬浮， 得到 20份正常细胞悬 液样品和 30份肿瘤细胞悬液样品，各细胞悬液样品的浓度在 104~109个细胞 /mL的范 围。
向每份样品中加入下式的化合物， 以使该化合物在各样品中的终浓度为 6μΜ
每份样品在 37°C培养箱中孵育 0.5小时， 然后在 4°C冰箱中继续孵育 0.5小时。 孵育结束后将样品离心， 去除染料溶液， 用 pH6的磷酸钠-磷酸氢钠缓冲液洗涤 细胞， 并制备 103~106个细胞 /ml的单细胞悬液。
用流式细胞仪检测各样品在 564-606nm处的荧光信号，获得染色阳性细胞占细胞 总数的比例， 即染色率。
对通过以上方式获得的 50份细胞样品的染色率进行统计分析，结果如图 3所示。 参照图 3， 可以清楚地看出所有结肠癌样品染色率大于 85%， 所有对照样品的染 色率均小于 40%， 因此，采用本实施例的诊断方式可以很容易地将正常细胞和结肠癌 诱导的肿瘤细胞分开。
实施例 10
采用与实施例 9相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检测， 区别在于， 所使用化合物为：
该化合物在各样品中的终浓度为 2μΜ， 孵育温度和时间为 37°C下 4小时， 4°C下 4小时， 用流式细胞仪检测各样品 670nm以上的荧光信号， 获得各样品的染色率。
检测结果:所有结肠癌样品染色率大于 75%，所有对照样品的染色率均小于 35%， 因此，采用本实施例的诊断方式可以很容易地将正常细胞和结肠癌诱导的肿瘤细胞分 开。
实施例 11
采用与实施例 9相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检测，
该化合物在各样品中的终浓度为： 50μΜ， 孵育温度和时间为 37°C下 10分钟， 用流式细胞仪检测各样品 670nm以上的荧光信号， 获得各样品的染色率。
检测结果:所有结肠癌样品染色率大于 90%，所有对照样品的染色率均小于 40%， 因此，采用本实施例的诊断方式可以很容易地将正常细胞和结肠癌诱导的肿瘤细胞分 开。
实施例 12
采用与实施例 9相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检领
检测结果:所有结肠癌样品染色率大于 55%，所有对照样品的染色率均小于 20%， 因此，采用本实施例的诊断方式可以很容易地将正常细胞和结肠癌诱导的肿瘤细胞分 开。
实施例 13
采用与实施例 9相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检测， 区别 ：
检测结果:所有结肠癌样品染色率大于 85%，所有对照样品的染色率均小于 40%， 因此，采用本实施例的诊断方式可以很容易地将正常细胞和结肠癌诱导的肿瘤细胞分 开。
实施例 14
培养结肠癌细胞 (GEO)。
培养正常细胞系， 包括仓鼠卵巢细胞 （CHO)， 鼠胚胎成纤维细胞 (NIH3T3), 人 肺成纤维细胞 (HLF)， 人血管平滑肌细胞 (HUMC)， 人皮肤细胞 (HaCaT), 将肿瘤细胞 株和正常细胞系制成单细胞悬液， 用 pH8的柠檬酸-柠檬酸钾缓冲液洗涤、 悬浮， 得 到正常细胞悬液样品和肿瘤细胞悬液样品， 各细胞悬液样品的浓度在 ιο4~ιο9个细胞
/mL的范围。
用流式细胞仪检测各单细胞悬液在 564-606nm处的荧光信号，获得染色阳性细胞 占各样品细胞总数的比例， 即染色率。
对通过以上方式重复 30次获得的 30份体外培养细胞的染色率进行统计分析，结
果如图 4所示。
参照图 4， 可以清楚地看出所有结肠癌细胞样品染色率大于 40%， 所有正常肺细 胞样品的染色率均小于 25%， 因此，采用本实施例的诊断方式可以很容易地将正常肺 细胞和肺肿瘤细胞分开。
实施例 15
采用与实施例 14相同的步骤对结肠癌细胞和正常细胞株进行检测， 区别在于， 所使
检测结果：所有结肠癌细胞样品染色率大于 45%，所有正常肺细胞样品的染色率 均小于 25%， 因此，采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细 胞分开。
实施例 16
采用与实施例 14相同的步骤对结肠癌细胞和正常细胞株进行检测， 区别在于， 所使用化合物为：
检测结果：所有结肠癌细胞样品染色率大于 90%，所有正常肺细胞样品的染色率 均小于 35%， 因此，采用本实施例的诊断方式可以很容易地将正常肺细胞和肺肿瘤细 胞分开。
实施例 17
饲养 50只裸鼠， 20只作为空白对照， 30只接种乳腺癌细胞（MCF-7)于裸鼠体 内， 待肿瘤长成后， 从接种 MCF-7的裸鼠取肿瘤组织， 空白对照组直接取乳腺及附 近的肌肉细胞，制成单细胞悬液，用 pH值为 6的磷酸钠-磷酸氢钠缓冲液洗涤、悬浮， 得到 20份浓度为 104~109个细胞 /mL的正常细胞悬液样品和 30份浓度为 104~109个 细胞 /mL的肿瘤细胞悬液样品。
度为 0.5μΜ
将每份样品在 37°C的培养箱中孵育 1小时， 然后在 4°C冰箱中继续孵育 1小时。 孵育结束后将样品离心， 去除染料溶液，用 pH值为 6的磷酸钠-磷酸氢钠缓冲液 洗涤细胞， 并重新悬浮得到 103~106个细胞 /ml的单细胞悬液。
用流式细胞仪检测细胞在 564-606nm处的荧光信号， 获得各样品的染色率。 对通过以上方式获得的 50份细胞样品的染色率进行统计分析，结果如图 5所示。 参照图 5， 可以清楚地看出所有的乳腺癌样品染色率大于 95%， 所有对照样品的 染色率都小于 25%，从而，采用本实施例的诊断方式可以很容易地将正常细胞和乳腺 癌诱导的肿瘤细胞分开。
实施例 18
采用与实施例 17相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检测， 区别在于， 所使用化合物为：
该化合物在样品中的浓度为 50μΜ， 孵育温度和时间为 37°C下 15 分钟， 4°C下 15分钟， 然后用流式细胞仪样品 670nm以上的荧光信号， 获得各样品的染色率。
检测结果：所有的乳腺癌样品的染色率都在 90%以上，所有的对照样品的染色率 都在 20%以下。
实施例 19
采用与实施例 17相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检测， 区别
检测结果：所有的乳腺癌样品的染色率都在 80%以上，所有的对照样品的染色率 都在 25%以下。
实施例 20
采用与实施例 17相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检测， 区别在于， 所使用的化合物为：
检测结果： 所有的乳腺癌样品的染色率都在 70%以上，所有的对照样品的染色率 都在 25%以下。
实施例 21
采用与实施例 17相同的步骤对以上 50只裸鼠的肿瘤细胞和正常细胞进行检测， 区别
实施例 22
培养人乳腺癌细胞 (MCF-7)。
培养正常细胞系， 包括仓鼠卵巢细胞 （CHO)， 鼠胚胎成纤维细胞 (NIH3T3), 人 肺成纤维细胞 (HLF)， 人血管平滑肌细胞 (HUMC)， 人皮肤细胞 (HaCaT), 用 pH8的巴 比妥钠 -盐酸缓冲液洗涤、悬浮所培养的乳腺癌细胞株和正常细胞系制成 104~109个细 胞 /mL的单细胞悬液， 得到正常细胞样品和乳腺癌细胞样品。
0.5μΜ，
每份样品在 37°C培养箱中孵育 4小时， 然后在 4°C冰箱中继续孵育 4小时。 孵育结束后将样品离心， 去除染料溶液， 用 pH8的巴比妥钠 -盐酸缓冲液洗涤细 胞， 制备 103~106个细胞 /ml的单细胞悬液。
用流式细胞仪检测细胞在 564-606nm 处的荧光信号， 获得染色阳性细胞团的比 例， 即染色率。
对通过以上方式重复 30次获得的 30份体外培养细胞的染色率进行统计分析，结 果如图 6所示。
参照图 6， 可以清楚地看出所有乳腺癌细胞样品染色率大于 40%， 所有正常乳腺 细胞样品的染色率均小于 25%， 因此，采用本实施例的诊断方式可以很容易地将正常 乳腺细胞和乳腺癌细胞区分开。
实施例 23
采用与实施例 22相同的步骤对乳腺癌细胞和正常乳腺细胞株进行检测， 区别在 于， 所使用化合物为：
该化合物在各样品中的终浓度为 200μΜ，孵育温度和时间为 37°C下 7.5小时， 4°C 下 0.5小时，用流式细胞仪检测个样品 670nm以上的荧光信号，获得各样品的染色率。
检测结果：所有乳腺癌细胞样品染色率大于 45%，所有正常乳腺细胞样品的染色 率均小于 25%。
实施例 24
采用与实施例 22相同的步骤对乳腺癌细胞和正常细胞株进行检测， 区别在于， 所使
该化合物在各样品中的终浓度为 1μΜ， 孵育温度和时间为 37°C下 2小时， 4°C下
2小时， 用流式细胞仪检测各样品 564-606nm处的荧光信号， 获得各样品的染色率。
检测结果：所有乳腺癌细胞样品染色率大于 35%，所有正常乳腺细胞样品的染色 率均小于 20%。
通过以上非限定性的实施例可以清楚地看出，使用本发明的系统和方法可以方便 地区分癌细胞和正常的组织细胞， 从而可以极大地为癌症的诊断提供便利。而且本发
明的方法的准确率极高， 所使用的试剂、 设备也容易获得， 操作简单。
需要说明的是， 本发明实施例中所使用的流式细胞仪为 Becton, Dickinson and CompanyCBD公司）生产的 FACSCalibur型号的流式细胞仪，其荧光激发波长为 488nm 和 688nm，但是在本发明中对流式细胞仪的型号并不做任何限定， 只要能够实施本发 明的方法即可； 在本发明的方法中对所各细胞悬液样品的浓度并不做特别的限定， 只 要能够满足流式细胞仪的检测要求即可，本领域技术人员可以根据需要来确定细胞悬 液的浓度。
虽然已经以具体实施例的方式描述了本发明，但是对于本领域技术人员来说明显 的是，在不脱离所附权利要求书所限定的本发明的精神和范围的情况下，可以对本发 明进行各种变化和修改， 这些变化和修改同样包括在本发明的范围内。
Claims
1. 一种诊断癌症的方法， 所述方法包括以下步骤：
( 1 ) 取得受试者的病灶组织样品， 并用 pH值为 6~8的缓冲液将取得的组 织样品制备成浓度为 104~109个细胞 /mL的单细胞悬液；
( 2 ) 向步骤（1 ) 中获得的单细胞悬液中加入式 I的化合物以使单细胞悬液 中式 I的化合物浓度为 0.5μΜ至 200μΜ， 并将获得的单细胞悬液先在 25-40 °C下 孵育 10 至 8小时， 然后在 2-8 °C下孵育 10分钟至 8小时；
( 3 ) 收集孵育后的细胞， 用 pH值为 6~8的缓冲液洗涤， 并重新悬浮， 得 到浓度为 103~106个细胞 /ml的单细胞悬液；
( 4 ) 用流式细胞仪在 515nm~650nm或 670nm以上波长范围内对步骤 （3 ) 中获得的细胞悬液的荧光信号进行检测， 计算细胞的染色率， 所述染色率为有荧 光信号产生的细胞占细胞总数的比例； 当染色率大于 20%， 优选大于 40%， 更 优选大于 60%， 进一步优选大于 80%时， 则可认定受试者患有癌症；
其中： 1^为 6的烷基、 苯基、 烷基取代的苯基； R2、 R3、 和 R5独立 地选自 H或 d-C6的烷基， 或者 R2和 R3与它们所连接的碳原子一起形成 5元至 7元的环结构， 或者 和 R5与它们所连接的碳原子一起形成 5元至 7元的环结 构； 和 R7为 d-C6烷基或者磺酸基取代的 d-C6烷基； Y为反离子， 根据 和 R7所带电荷的不同而不同， 若 和 R7为烷基， 则 Y为卤素阴离子； 若 Re 和 R7中只有一个带有磺酸根， 则无需 Y作为反离子； 若 和 R7均带有磺酸根， 则 Y为三乙胺阳离子； Xi、 X2独立地选自 C、 0、 S、 Se或 Te。
2. 如权利要求 1 所述的方法， 其中所述缓冲液为含有一价金属离子的缓冲 液。
3. 如权利要求 1 所述的方法， 其中所述缓冲液为含有钾离子或钠离子的缓 冲液。
4. 如权利要求 1所述的方法，其中所述缓冲液选自磷酸钠 -磷酸氢钠缓冲液、 磷酸钾 -磷酸氢钾缓冲液、 巴比妥钠-盐酸缓冲液、 巴比妥钾-盐酸缓冲液、柠檬酸
-柠檬酸钠缓冲液或柠檬酸 -柠檬酸钾缓冲液。
5. 如权利要求 1所述的方法， 其中所述 d-C6的烷基选自甲基、 乙基、 正丙 基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基或异己基。
6. 如权利要求 1所述的方法， 其中 选自甲基、 乙基、 正丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基、 异己基、 苯基、 甲基苯基或 二甲基苯基。
7. 如权利要求 1所述的方法，其中 R2、 R3、 和 R5独立地选自甲基、乙基、 正丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基或异己基。
8. 如权利要求 1所述的方法，其中所述 5元至 7元环结构为含有或不含有 N 或 S原子的饱和或不饱和环结构。
9. 如权利要求 1所述的方法， 其中 Y选自氟、 氯、 溴、 碘阴离子或三乙胺 阳离子。
10. 如权利要求 1至 9中任一项所述的方法， 其中所述癌症为肺癌、 结肠癌 或乳腺癌。
11. 一种用于诊断癌症的试剂盒和系统， 所述试剂盒包括试剂 I和试剂 II， 所述系统包括所述试剂盒和流式细胞仪； 其中所述试剂 I为 pH值为 6~8的缓冲 液， 试剂 II为下式 I所示的化合物：
其中： 1^为 6的烷基、 苯基、 烷基取代的苯基； R2、 R3、 和 R5独立 地选自 H或 d-C6的烷基， 或者 R2和 R3与它们所连接的碳原子一起形成 5元至 7元的环结构， 或者 和 R5与它们所连接的碳原子一起形成 5元至 7元的环结 构； 和 R7为 d-C6烷基或者磺酸基取代的 d-C6烷基； Y为反离子， 根据 和 R7所带电荷的不同而不同， 若 和 R7为烷基， 则 Y为卤素阴离子； 若 Re 和 R7中只有一个带有磺酸根， 则无需 Y作为反离子； 若 和 R7均带有磺酸根， 则 Y为三乙胺阳离子； Xi、 X2独立地选自 C、 0、 S、 Se或 Te。
12. 如权利要求 11 所述的试剂盒和系统， 其中所述缓冲液为含有一价金属 离子的缓冲液。
13. 如权利要求 11 所述的试剂盒和系统， 其中所述缓冲液为含有钾离子或 钠离子的缓冲液。
14. 如权利要求 11所述的试剂盒和系统， 其中所述缓冲液选自磷酸钠 -磷酸 氢钠缓冲液、磷酸钾 -磷酸氢钾缓冲液、 巴比妥钠-盐酸缓冲液、 巴比妥钾-盐酸缓 冲液、 柠檬酸 -柠檬酸钠缓冲液或柠檬酸 -柠檬酸钾缓冲液。
15. 如权利要求 11所述的试剂盒和系统， 其中所述 d-C6的烷基选自甲基、 乙基、 正丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基或异 己基。
16. 如权利要求 11所述的试剂盒和系统， 其中 选自甲基、 乙基、正丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己基、 异己基、 苯基、 甲 基苯基或二甲基苯基。
17. 如权利要求 11所述的试剂盒和系统， 其中 R2、 R3、 和 R5独立地选自 甲基、 乙基、 正丙基、 异丙基、 正丁基、 异丁基、 叔丁基、 戊基、 异戊基、 正己 基或异己基。
18. 如权利要求 11所述的试剂盒和系统， 其中所述 5元至 7元环结构为含 有或不含有 N或 S原子的饱和或不饱和环结构。
19. 如权利要求 11所述的试剂盒和系统， 其中 Y选自氟、 氯、 溴、 碘阴离 子或三乙胺阳离子。
20. 如权利要求 11至 19中任一项所述的试剂盒和系统，其中述癌症为肺癌、 结肠癌或乳腺癌。
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| CN201210164349.XA CN102703569B (zh) | 2012-05-24 | 2012-05-24 | 菁染料的新用途 |
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| EP0417941A2 (en) * | 1989-08-30 | 1991-03-20 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Use of a cyanine dye in an antitumor agent |
| CN102268191A (zh) * | 2010-06-06 | 2011-12-07 | 史春梦 | 七甲川吲哚花菁染料及其合成方法和应用 |
| CN102703569A (zh) * | 2012-05-24 | 2012-10-03 | 中国科学院化学研究所 | 菁染料的新用途 |
| CN102706788A (zh) * | 2012-05-24 | 2012-10-03 | 中国科学院化学研究所 | 用于检测乳腺癌的系统 |
| CN102706787A (zh) * | 2012-05-24 | 2012-10-03 | 中国科学院化学研究所 | 菁染料的新用途 |
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| EP0417941A2 (en) * | 1989-08-30 | 1991-03-20 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Use of a cyanine dye in an antitumor agent |
| CN102268191A (zh) * | 2010-06-06 | 2011-12-07 | 史春梦 | 七甲川吲哚花菁染料及其合成方法和应用 |
| CN102703569A (zh) * | 2012-05-24 | 2012-10-03 | 中国科学院化学研究所 | 菁染料的新用途 |
| CN102706788A (zh) * | 2012-05-24 | 2012-10-03 | 中国科学院化学研究所 | 用于检测乳腺癌的系统 |
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