CN106117270A - A kind of annular metal iridium complex and preparation method thereof and the application as protein staining agent - Google Patents
A kind of annular metal iridium complex and preparation method thereof and the application as protein staining agent Download PDFInfo
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- CN106117270A CN106117270A CN201610364682.3A CN201610364682A CN106117270A CN 106117270 A CN106117270 A CN 106117270A CN 201610364682 A CN201610364682 A CN 201610364682A CN 106117270 A CN106117270 A CN 106117270A
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- iridium complex
- annular metal
- metal iridium
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- 229910052741 iridium Inorganic materials 0.000 title claims abstract description 73
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 title claims abstract description 73
- 229910052751 metal Inorganic materials 0.000 title claims abstract description 72
- 239000002184 metal Substances 0.000 title claims abstract description 72
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 72
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 72
- 238000010186 staining Methods 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 238000010668 complexation reaction Methods 0.000 title abstract description 4
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 31
- 239000003446 ligand Substances 0.000 claims abstract description 28
- 238000004043 dyeing Methods 0.000 claims abstract description 26
- 125000002091 cationic group Chemical group 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 43
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- 150000001875 compounds Chemical class 0.000 claims description 35
- 239000003292 glue Substances 0.000 claims description 35
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethyl sulfoxide Natural products CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 17
- 229910052801 chlorine Inorganic materials 0.000 claims description 15
- 239000000460 chlorine Substances 0.000 claims description 15
- 229910052736 halogen Inorganic materials 0.000 claims description 15
- 150000002367 halogens Chemical group 0.000 claims description 15
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 10
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 10
- 229910052723 transition metal Inorganic materials 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 150000003624 transition metals Chemical class 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 8
- -1 amino, hydroxyl Chemical group 0.000 claims description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 5
- CUJRVFIICFDLGR-UHFFFAOYSA-N acetylacetonate Chemical compound CC(=O)[CH-]C(C)=O CUJRVFIICFDLGR-UHFFFAOYSA-N 0.000 claims description 4
- PSLMOSLVUSXMDQ-UHFFFAOYSA-N iridium;pentane-2,4-dione Chemical compound [Ir].CC(=O)CC(C)=O PSLMOSLVUSXMDQ-UHFFFAOYSA-N 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 claims description 3
- 125000004429 atom Chemical group 0.000 claims description 3
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 238000007447 staining method Methods 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims 6
- 239000001257 hydrogen Substances 0.000 claims 6
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims 6
- 239000002253 acid Substances 0.000 claims 2
- 239000006184 cosolvent Substances 0.000 claims 2
- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical group O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 claims 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000007547 defect Effects 0.000 abstract description 2
- 238000003384 imaging method Methods 0.000 description 20
- 238000000034 method Methods 0.000 description 13
- 239000002904 solvent Substances 0.000 description 10
- 230000005284 excitation Effects 0.000 description 9
- 150000002503 iridium Chemical class 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000010413 mother solution Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 5
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 4
- 229910052794 bromium Inorganic materials 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 229910052740 iodine Inorganic materials 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 4
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- ZNQVEEAIQZEUHB-UHFFFAOYSA-N 2-ethoxyethanol Chemical class CCOCCO ZNQVEEAIQZEUHB-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 150000001335 aliphatic alkanes Chemical group 0.000 description 2
- 125000001309 chloro group Chemical group Cl* 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 229940125904 compound 1 Drugs 0.000 description 2
- 150000004696 coordination complex Chemical class 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000004210 ether based solvent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002795 fluorescence method Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 150000002504 iridium compounds Chemical class 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 229910001961 silver nitrate Inorganic materials 0.000 description 2
- 229910021135 KPF6 Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000012455 bioassay technique Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0033—Iridium compounds
-
- 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
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- C09K2211/18—Metal complexes
- C09K2211/185—Metal complexes of the platinum group, i.e. Os, Ir, Pt, Ru, Rh or Pd
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Materials Engineering (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of annular metal iridium complex and preparation method thereof and the application as protein staining agent, annular metal iridium complex includes C^N bidentate ligand and/or N^N bidentate ligand structure, it belongs to cationic or electric neutrality luminescent metal complexes, has higher quantum efficiency；Dyeing during as protein staining agent highly sensitive, can reach ng level, dyeing, without rinse step, is more saved the time；Solving prior art needs rinsing, the low defect of sensitivity and annular metal iridium complex preparation method disclosed by the invention simple, and stability is high.
Description
Technical field
The invention belongs to bioassay technique field, be specifically related to a kind of annular metal iridium complex and preparation method thereof and work
Application for protein staining agent.
Background technology
Electrophoresis is widely used in the biological relevant field such as analysis of protein, drug development as a kind of important technology.
Utilize electrophoresis means can by protein according to the difference of molecular weight at SDS-polyacrylamide gel slab
(SDS-PAGE) different bands is isolated on.The visualization of these protein bands be by subsequent protein analyze premise and
Basis.
Albumen method for visualizing includes colorimetry and fluorescence method two kinds at present.Dyestuff used by colorimetry has silver nitrate and examines
Maas light blue etc., but silver nitrate and protein staining are by irreversible chemical bonds, are unfavorable for follow-up protein
Analyze, the wayward dyeing condition of argentation simultaneously, higher to the operation level requirement of technical staff；Coomassie brilliant blue sensitive
Spend poor.Dyestuff used by fluorescence method includes little molecule and the big class of metal complex two, wherein metal complex luminescent dye with
Its stability is higher, and luminous efficiency relatively advantages of higher occupies critical role in protein staining agent market；But existing metal is joined
Compound less stable, dyeing sensitivity is on the low side, about at hundreds of nanogram, and needs the rinse step of complexity.
Summary of the invention
It is an object of the invention to provide the protein staining agent that a class is novel.
To achieve the above object of the invention, the technical solution used in the present invention is: a kind of annular metal iridium complex, belong to sun from
Subtype or electroneutral complex, central metal atom is iridium, can be the novel protein staining agent of a class.Concrete, it is changed
Structural formula is as follows:
Or。
Wherein C^N bidentate ligand is main part, its structural formula can be following any one:
Wherein R can be-H, alkyl (-CH3, -CH2CH3), halogen, amino (-NH2), nitro (-NO2), sulfonic group (-SO3)
Deng；
N^N bidentate ligand is assistant ligand, and its structure can be following any one:
Wherein R` can be-H, alkyl (-CH3, -CH2CH3), halogen (Cl, Br, I), hydroxyl (-OH), carboxyl (-COOH), ammonia
Base (-NH2), nitro (-NO2), sulfonic group (-SO3) etc.；
X is to ion, can be halogen (Cl, Br, I), hexafluoro-phosphate radical (PF6) etc..
In technique scheme, alkyl is-CH3Or-CH2CH3；Halogen is Cl, Br or I.
Preferably, the chemical structural formula of described annular metal iridium complex is as follows:
。
The invention also discloses the application as protein staining agent of the above-mentioned annular metal iridium complex.
The invention also discloses the application in protein staining of the above-mentioned annular metal iridium complex.
The protein staining process that the annular metal iridium complex of the present invention is used in gel electrophoresis technology as protein staining agent；
Therefore the present invention further discloses the application when gel electrophoresis is tested in protein staining of the above-mentioned annular metal iridium complex.
The annular metal iridium complex of the present invention is when protein staining, and the concentration of annular metal iridium complex is 0. 1 micromoles
Between 10 mMs, that optimum is 1 micromole；Annular metal iridium complex is 3-20 hour with the common incubation time of albumen,
Excellent is 6 hours；Need the hydrotropy by means of organic solvent, such as dimethyl sulfoxide (DMSO), N, N '-dimethyl Methanamide, first
Alcohol etc..And the annular metal iridium complex of the present invention is when protein staining, protein staining agent is washed without de-in dyeing course,
Directly being taken out by glue and be placed in imaging in gel imaging system, sensitivity is the highest.
The invention also discloses the preparation method of above-mentioned annular metal iridium complex, comprise the following steps, with iridous chloride three
Crystalline hydrate compound and C^N bidentate ligand are that raw material prepares transition metal chlorine endo compound, then with transition metal chlorine bridge
Compound, N^N bidentate ligand compound are that annular metal iridium complex prepared by raw material；Or with acetyl acetone iridium Ir (acac)3With C
^N bidentate ligand is that annular metal iridium complex prepared by raw material；Or join with C^N bis-tooth with iridous chloride three crystalline hydrate compound
Body is that transition metal chlorine endo compound prepared by raw material；Then it is former with transition metal chlorine endo compound, N^N bidentate ligand compound
Chloride ion complex of iridium prepared by material, then reacts with bromide, iodide or hexafluorophosphoric acid compound and obtain Cyclometalated iridium and coordinate
Thing.Bromide, iodide or hexafluorophosphoric acid compound can be NaBr, KI, KPF respectively6。
In technique scheme, after reacting 24 hours in ethylene glycol ethyl ethers ether solvents, obtain transition metal chlorine endo compound,
Further react, purify and obtain final ion-type Luminous Ring metal iridium compound；With acetyl acetone iridium Ir (acac)3With
C^N bidentate ligand is raw material, reacting by heating 24 hours, obtains electroneutral Luminous Ring metal iridium complex through column chromatography for separation.
The invention also discloses and utilize annular metal iridium complex to carry out the concrete steps of protein staining, albumen as stain
After SDS-PAGE, fix in ethanol/acetic acid and obtain glue；Glue is inserted in annular metal iridium complex solution after rinsing, complete
Become dyeing；It is specifically as follows by protein sample after SDS-PAGE, is placed in the ethanol of 30%, liquid was fixed by 10% acetic acid and obtains
Glue；Glue is placed in 20% ethanol rinsing 3 times, each 30 minutes；By protein staining agent iridium complexes from mother solution (with
DMSO is solvent), it is diluted to 1 micromole with pure water, be then placed in one dyeing 6 hours by glue, completes dyeing；Directly by the 3rd
Glue after step takes out and is placed in imaging in gel imaging system, selects 312nm as excitation wavelength, and 515nm is as launching wavelength.
The invention also discloses a kind of protein staining agent, its structural formula is:
Or；
Itself belonging to cationic or electroneutral complex, central metal atom is iridium.
Wherein C^N bidentate ligand is main part, its structural formula can be following any one:
Wherein R can be-H, alkane chain (-CH3, -CH2CH3), halogen, amino (-NH2), nitro (-NO2), sulfonic group (-
SO3) etc.；
N^N bidentate ligand is assistant ligand, and its structure can be following any one:
Wherein R` can be-H, alkane chain (-CH3, -CH2CH3), halogen, hydroxyl (-OH), carboxyl (-COOH), amino (-
NH2), nitro (-NO2), sulfonic group (-SO3) etc.；
X is to ion, can be halogen (Cl, Br, I), hexafluoro-phosphate radical (PF6) etc..
The present invention further discloses a kind of kinds of staining methods for protein, by albumen after SDS-PAGE, solid in ethanol/acetic acid
Surely glue is obtained；Glue is inserted in above-mentioned annular metal iridium complex solution after rinsing, completes dyeing.
Owing to technique scheme is used, present invention have the advantage that
1. the invention discloses a class Luminous Ring metal iridium complex, dye during as protein staining agent highly sensitive, Ke Yida
To ng level, dyeing, without rinse step, is more saved the time；Solve the defect that prior art needs to rinse, sensitivity is low.
Annular metal iridium complex preparation method the most disclosed by the invention is simple, and stability is high, and quantum efficiency is higher；For
During gel test, protein staining is effective, can reach ng level.
Accompanying drawing explanation
Fig. 1 is the fluorogram of gel-tape in embodiment three；
Fig. 2 is the fluorogram of gel-tape in embodiment four；
Fig. 3 is the fluorogram of gel-tape in embodiment five；
Fig. 4 is the fluorogram of gel-tape in embodiment six；
Fig. 5 is the fluorogram of gel-tape in embodiment seven；
Fig. 6 is the fluorogram of annular metal iridium complex in embodiment six；
Fig. 7 is the fluorogram of annular metal iridium complex in embodiment four；
Fig. 8 is the fluorogram of annular metal iridium complex in embodiment three.
Detailed description of the invention
The synthesis of embodiment one ion-type Luminous Ring metal iridium complex
First iridous chloride three crystalline hydrate compound and C^N bidentate ligand (compound 1) in ethylene glycol ethyl ethers ether solvents anti-
After answering 24 hours, obtain transition metal chlorine endo compound (compound 2), the most further chlorine endo compound is joined with N^N bis-tooth
Body compound (compound 3) reacts, and solvent is spin-dried for, and obtains final ion-type Luminous Ring metal iridium compound through column chromatography for separation
(compound 4).Productivity: 68%.Mass spectrum: experiment value [M-Cl]+655.1610, theoretical value [M-Cl]+ 655.1601。
C^N bidentate ligand, N^N bidentate ligand compound being replaced, the ion-type that can obtain multiple structure is luminous
Annular metal iridium complex.
Can also by chloride ion complex of iridium obtained above, then with NaBr, KI or KPF6Reaction obtain different to from
The annular metal iridium complex of son.
The preparation of embodiment two electric neutrality Luminous Ring metal iridium complex
By acetyl acetone iridium Ir (acac)3With C^N bidentate ligand compound 1 reacting by heating 24 hours, obtain through column chromatography for separation
To electroneutral Luminous Ring metal iridium complex 2.Productivity is 60%.Mass spectrum: experiment value [M+H]+655.1510, theoretical value [M
+H]+ 655.1599。
Being replaced by C^N bidentate ligand compound, the electric neutrality Luminous Ring metal iridium that can obtain multiple structure coordinates
Thing.
Embodiment three
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 37%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 3 times, each 30 minutes；
3, by protein staining agent iridium complexes from mother solution (with DMSO as solvent), be diluted to 1 micromole with pure water, then
Be placed in one dyeing 6 hours by glue；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.
The protein label of the commercialization after electrophoretic separation (is divided by 1 micromolar Luminous Ring metal iridium complex
Son amount is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4 KDa, 14.4 KDa) carry out
Dyeing, the fluorogram of gel-tape is accompanying drawing 1；Band in figure is followed successively by from a left side to protein content, 500ng, 250ng,
125ng, 62.5ng, 31.25ng。
Embodiment four
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 46%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 3 times, each 30 minutes；
3, by protein staining agent iridium complexes from mother solution (with DMSO as solvent), be diluted to 1 micromole with pure water, then
Be placed in one dyeing 6 hours by glue；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.
The protein label of the commercialization after electrophoretic separation (is divided by 1 micromolar Luminous Ring metal iridium complex
Son amount is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4 KDa, 14.4 KDa) carry out
Dyeing, the fluorogram of gel-tape is accompanying drawing 2；Band in figure is followed successively by from a left side to protein content, 500ng, 250ng,
125ng, 62.5ng, 31.25ng。
Embodiment five
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 23%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 3 times, each 30 minutes；
3, by protein staining agent iridium complexes from mother solution (with N, N '-dimethyl Methanamide is solvent), dilute with pure water
To 1 micromole, be then placed in one dyeing 6 hours by glue；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.
The protein label of the commercialization after electrophoretic separation (is divided by 1 micromolar Luminous Ring metal iridium complex
Son amount is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4 KDa, 14.4 KDa) carry out
Dyeing, the fluorogram of gel-tape is accompanying drawing 3；Band in figure is followed successively by from a left side to protein content, 500ng, 250ng,
125ng, 62.5ng, 31.25ng。
Embodiment six
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 28%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 3 times, each 30 minutes；
3, by protein staining agent iridium complexes from mother solution (with methanol as solvent), be diluted to 1 micromole with pure water, then
Be placed in one dyeing 6 hours by glue；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.
The protein label of the commercialization after electrophoretic separation (is divided by 1 micromolar Luminous Ring metal iridium complex
Son amount is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4 KDa, 14.4 KDa) carry out
Dyeing, the fluorogram of gel-tape is accompanying drawing 4；Band in figure is followed successively by from a left side to protein content, 500ng, 250ng,
125ng, 62.5ng, 31.25ng。
Embodiment seven
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 40%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 3 times, each 30 minutes；
3, by protein staining agent iridium complexes from mother solution (with DMSO as solvent), be diluted to 1 micromole with pure water, then
Be placed in one dyeing 6 hours by glue；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.
The protein label of the commercialization after electrophoretic separation (is divided by 1 micromolar Luminous Ring metal iridium complex
Son amount is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4 KDa, 14.4 KDa) carry out
Dyeing, the fluorogram of gel-tape is accompanying drawing 5；Band in figure is followed successively by from a left side to protein content, 500ng, 250ng,
125ng, 62.5ng, 31.25ng。
On the whole, the annular metal iridium complex of the present invention has higher quantum efficiency, sees accompanying drawing 6-8, can see
Going out, the annular metal iridium complex of the present invention is under fluorescence irradiates, and quantum efficiency is high；These complex of iridium are utilized to carry out protein staining
Effective, it is not necessary to carry out albumen rinse step, protein band is the most high-visible, and detection sensitivity, at 30 below ng, shows this
Class coordination compound has the biggest development and application prospect as protein staining agent.
Embodiment eight
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 52%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample (molecular weight is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4
KDa, 14.4 KDa) after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 2 times, each 40 minutes；
3, by protein staining agent iridium complexes from mother solution (with DMSO as solvent), be diluted to 0.1 micromole with pure water, so
After glue is placed in one dyeing 20 hours；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.The fluorescence results of gel-tape is it can be seen that minimum protein content is 58.21ng.
Embodiment nine
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 63%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample (molecular weight is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4
KDa, 14.4 KDa) after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 4 times, each 20 minutes；
3, by protein staining agent iridium complexes from mother solution (with methanol as solvent), be diluted to 10 mMs with pure water, so
After glue is placed in one dyeing 3 hours；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.The fluorescence results of gel-tape is it can be seen that minimum protein content is 56.33ng.
Embodiment ten
Protein staining agent structural formula used is:
The fluorescence quantum efficiency of this compound is 46%.
The annular metal iridium complex of the present invention being used for protein staining and does gel test, process is specific as follows:
1, by protein sample (molecular weight is followed successively by: 116KDa, 66.2 KDa, 45 KDa, 35 KDa, 25 KDa, 18.4
KDa, 14.4 KDa) after SDS-PAGE, it is placed in the ethanol of 30%, liquid fixed by 10% acetic acid；
2, glue is placed in 20% ethanol rinsing 3 times, each 30 minutes；
3, by protein staining agent iridium complexes from mother solution (with DMSO as solvent), be diluted to 2 micromoles with pure water, then
Be placed in one dyeing 8 hours by glue；
4, directly the glue taking-up after the 3rd step is placed in imaging in gel imaging system.312nm is as excitation wavelength in selection,
515nm is as launching wavelength.The fluorescence results of gel-tape is it can be seen that minimum protein content is 48.88ng.
Claims (10)
1. an annular metal iridium complex, it is characterised in that: described annular metal iridium complex is cationic or electric neutrality is joined
Compound, central metal atom is iridium.
Annular metal iridium complex the most according to claim 1, it is characterised in that: the chemical constitution of described annular metal iridium complex
As follows:
Or；
Wherein, X is to ion.
Annular metal iridium complex the most according to claim 2, it is characterised in that: the structural formula of C^N bidentate ligand is following knot
Any one in structure formula:
Wherein R is hydrogen, alkyl, halogen, amino, nitro or sulfonic group；
The structural formula of N^N bidentate ligand is any one in following structural formula:
Wherein R` is hydrogen, alkyl, halogen, amino, hydroxyl, carboxyl, nitro or sulfonic group；
X is halogen or hexafluorophosphoricacid acid ions.
Annular metal iridium complex the most according to claim 3, it is characterised in that: the structural formula of described annular metal iridium complex is
One in following chemical structural formula:
。
5. the application in protein staining of the annular metal iridium complex described in claim 1.
Application the most according to claim 5, it is characterised in that: the concentration of annular metal iridium complex be 0.1 mol～
10mmol；Dyeing time is 3～20 hours；Cosolvent is organic solvent.
Application the most according to claim 6, it is characterised in that: the concentration of annular metal iridium complex is 1 mol；Dyeing time
It it is 6 hours；Cosolvent is dimethyl sulfoxide, N, N '-dimethyl Methanamide, methanol.
8. the preparation method of annular metal iridium complex described in claim 1, comprises the following steps, with iridous chloride three crystalline hydrate
Compound and C^N bidentate ligand are that raw material prepares transition metal chlorine endo compound, then with transition metal chlorine endo compound, N^N
Bidentate ligand compound is that annular metal iridium complex prepared by raw material；Or with acetyl acetone iridium Ir (acac)3With C^N bis-tooth
Part is that annular metal iridium complex prepared by raw material；Or it is former with iridous chloride three crystalline hydrate compound and C^N bidentate ligand
Transition metal chlorine endo compound prepared by material；Then prepare with transition metal chlorine endo compound, N^N bidentate ligand compound for raw material
Chloride ion complex of iridium, then react obtain annular metal iridium complex with bromide, iodide or hexafluorophosphoric acid compound；Described
The structural formula of C^N bidentate ligand is any one in following structural formula:
Wherein R is hydrogen, alkyl, halogen, amino, nitro or sulfonic group；
The structural formula of N^N bidentate ligand is any one in following structural formula:
Wherein R` is hydrogen, alkyl, halogen, amino, hydroxyl, carboxyl, nitro or sulfonic group.
9. a protein staining agent, its structural formula is:
Or；
The structural formula of C^N bidentate ligand is any one in following structural formula:
Wherein R is hydrogen, alkyl, halogen, amino, nitro or sulfonic group；
The structural formula of N^N bidentate ligand is any one in following structural formula:
Wherein R` is hydrogen, alkyl, halogen, amino, hydroxyl, carboxyl, nitro or sulfonic group；
X is halogen or hexafluorophosphoricacid acid ions.
10. utilize annular metal iridium complex described in claim 1 to carry out kinds of staining methods for protein, by albumen after SDS-PAGE,
Ethanol/acetic acid is fixed and obtains glue；Glue is inserted in annular metal iridium complex solution after rinsing, completes dyeing.
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| CN107245085A (en) * | 2017-07-11 | 2017-10-13 | 安徽大学 | Iridium complex with tubulin recognition function and preparation method and application thereof |
| CN107245085B (en) * | 2017-07-11 | 2019-05-31 | 安徽大学 | Iridium complex with tubulin recognition function and preparation method and application thereof |
| CN108707168A (en) * | 2018-04-13 | 2018-10-26 | 苏州科技大学 | Annular metal iridium complex containing sulfone and the organic electroluminescence device based on the complex |
| CN108707168B (en) * | 2018-04-13 | 2020-06-26 | 苏州科技大学 | Sulfone ring-containing metal iridium complex and organic electroluminescent device based on complex |
| CN110320086A (en) * | 2019-07-09 | 2019-10-11 | 上海宝藤生物医药科技股份有限公司 | Lipoprotein electrophoresis staining solution and preparation method and application thereof |
| CN110320086B (en) * | 2019-07-09 | 2020-04-14 | 上海宝藤生物医药科技股份有限公司 | Lipoprotein electrophoresis staining solution and preparation method and application thereof |
| CN115466293A (en) * | 2022-09-30 | 2022-12-13 | 苏州科技大学 | Aggregation-induced emission complex and preparation method thereof, complex system, application and application method |
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