CN102798561A - Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagent for cell nucleolus - Google Patents
Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagent for cell nucleolus Download PDFInfo
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- CN102798561A CN102798561A CN2012102772534A CN201210277253A CN102798561A CN 102798561 A CN102798561 A CN 102798561A CN 2012102772534 A CN2012102772534 A CN 2012102772534A CN 201210277253 A CN201210277253 A CN 201210277253A CN 102798561 A CN102798561 A CN 102798561A
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Abstract
Application of gold-silver mixed-metal cluster compound in preparing fluorescence staining reagents for cell nucleoli relates to a fluorescence staining reagent. The invention provides an application of the gold-silver mixed-metal cluster compound which has excellent specific staining effect to cell nucleolus area quickly and stably, in preparing fluorescence staining reagents. The gold-silver mixed-metal cluster compound [Au6Ag2(C)(dppy)6](BF4)4 is synthesized using the method in the document: Jian-Hua Jia, Quan-Ming Wang; J. Am. Chem. Soc. 2009, 131, 16634-16635. The dppy is diphenyl-2-pyridylphosphine. The gold-silver mixed-metal cluster compound has fluorescence staining effect to cell nucleolus area, wherein the cell nucleolus fluorescence staining performance of the gold-silver mixed-metal cluster compound reaches or exceeds that of existing like products, so the compound can be used for preparing fluorescence staining reagents for cell nucleoli.
Description
Technical field
The present invention relates to a kind of fluorescent dye reagent, especially relate to the application of the different metal cluster compound of a kind of gold and silver in preparation entoblast fluorescent dye reagent.
Background technology
Kernel (nucleolus) is the most significant structure in the eukaryotic interphase nucleus, and it is the spherical corpusculum of single or a plurality of homogeneous.The size of kernel, shape and number change with kind, cell type and the cellular metabolism state of biology.Kernel in eukaryotic has very important function, and it is that rRNA is synthetic, the assembling place of processing and ribosomal subunit.Therefore to nucleolar structure, dynamically and the research of function, be not merely early stage cytologist and pay close attention to, and since finding the critical function of kernel the sixties in last century, enjoyed each association area researcher's great attention always yet.The structure observation of pair cell kernel is carried out through Electronic Speculum the earliest and since need to biological sample dewater, processing such as dyeing, embedding, viewed is the structure of a static state, has therefore limited the further research to the physiological function of kernel.
The visual detection approach that fluorescence imaging is a sensitivity, non-intrusion type, expense is cheap relatively, its resolution can reach hundred nanometers, can realize the living imaging from the cell to the animal, is an important method of present modern scientific research.The regional fluorescent dye of cell is very important work.Yet there is following shortcoming in the cell fluorescence label probe at present: at first, light stability is poor, light takes place rapidly under the exciting light Continuous irradiation drift.Secondly, cell is prone to take place optical damage, and particularly under the situation of fluorophore existence, the free radical that produces when photobleaching takes place can cause damage by the pair cell sample.Therefore, the stable cell fluorescence staining reagent of invention light is very important work.
Summary of the invention
The object of the present invention is to provide a kind of application of the different metal cluster compound of gold and silver in preparation entoblast fluorescent dye reagent that the specific stain excellent effect is carried out in quick, stable pair cell kernel zone that have.
Different metal cluster compound [the Au6Ag of said gold and silver
2(C) (dppy)
6] (BF
4)
4Use document: Jian-Hua Jia, Quan-Ming Wang; J.Am.Chem.Soc.2009,131, the method for 16634-16635 is synthetic.Dppy=diphenylphosphino-2-pyridine.
The different metal cluster compound pair cell of said gold and silver kernel zone has the fluorescent dye effect, and its entoblast dyeability meets or exceeds existing known like product level, can be used for preparing entoblast fluorescent dye reagent.
The present invention considers that different metal cluster compound has abundant structure-activity relationship, and through structure optimization and screening, the different metal cluster compound of the gold and silver that obtains has the excellent effect that specific stain is carried out in quick, stable pair cell kernel zone.
The different metal cluster compound of gold and silver according to the invention is that a fluid-like state is sent out high light and the high compound of stability.Test cell line shows; The present invention designs the different metal cluster compound of synthetic gold and silver and can realize specific stain is carried out in HeLa entoblast zone; Light stability is good, and imaging is stable, under identical exciting light prolonged exposure; The fluorescently-labeled cell of the present invention has very strong anti-optical quenching ability, is difficult for taking place photobleaching.Adopt the inventive method labeled cell convenient and swift, the fluorescence signal mark time is long, and wide accommodation, and the method and the kit of invention can be used for living cells, also can be used for the fixed cell mark.This series cluster compound, simple in structure, synthesis technique is simple and easy and efficient is high, with low cost, in biologic pharmacological science, has boundless application prospect.
Description of drawings
Fig. 1 is the laser co-focusing image of the different metal cluster compound of gold and silver in the HeLa cell.In Fig. 1, scale is 10 μ m; A is the different metal cluster compound of gold and silver, and b is a light field, and c is stack.
Fig. 2 is that the different metal cluster compound of gold and silver is at intracellular emission spectrum.In Fig. 2, horizontal ordinate is wavelength Wavelength (nm), and ordinate is spectral intensity Luminescence intensity (a.u).
Fig. 3 is for the different metal cluster compound of gold and silver and dye the common location fluorescence imaging figure of kernel reagent Syto Green RNA-select in the HeLa cell.In Fig. 3, scale is 10 μ m; A is Syto Green RNA-select, and b is the different metal cluster compound of gold and silver, and c is a light field, and d is stack.
Fig. 4 is for the different metal cluster compound of gold and silver and dye kernel reagent Syto Green RNA Select at intracellular emission spectrum.In Fig. 4, horizontal ordinate is wavelength Wavelength (nm), and ordinate is spectral intensity Luminescence intensity (a.u); A is for dying kernel reagent Syto Green RNA Select, and b is the different metal cluster compound of gold and silver.
Fig. 5 be different metal cluster compound of gold and silver and organic dyestuff DAPI light stability relatively (405nm laser prolonged exposure, different time points collection are 0,200s, 400s's forms images through the HeLe cell confocal fluorescent of the different metal cluster compound of gold and silver with DAPI dyeing.In Fig. 5, DAPI is an organic dyestuff, and J1 is the different metal cluster compound of gold and silver.
Fig. 6 is the light stability photobleaching rate curve relatively of different metal cluster compound of gold and silver and organic dyestuff DAPI.In Fig. 6, horizontal ordinate is time T ime (s), and ordinate is intensity I ntensity (a.u); Curve a is that (460 ± 20nm), curve is the different metal cluster compound (570 ± 20nm) of gold and silver to DAPI.
Embodiment
The entoblast fluorescent dye of the different metal cluster compound of gold and silver:
1) human cervical carcinoma cell strain HeLa (available from Chinese Academy of Sciences's Shanghai cell bank), in containing the DMEM nutrient solution of 10% hyclone, 37 ℃, 5%CO
2, cultivate under the saturated humidity condition.Changed nutrient solution, went down to posterity once in per 3～4 days in per two days.
2) processing of cellular incubation slide.The cover glass of Φ 14mm is washed, put into sulfuric acid-potassium dichromate solution and soak 24h, the distilled water washing, bubble is gone into absolute ethyl alcohol, and is subsequent use.
3) preparation of cell climbing sheet.Cover glass is tiled in the double dish of Φ 35mm, with 1 * 10
5Cell/mL inoculation, 37 ℃, 5%CO
2, put incubator and cultivate.
4) treat cell attachment, with phosphate buffer (PBS) washing 3 times, adding final concentration is the different metal cluster compound solution of 10 μ M gold and silver incubated at room 10min, sucking-off solution subsequently, PBS washed cell 3 times, each 3～5min.
5) with 405nm laser as exciting light (FV-LD40 laser instrument, Olympus, 25mW, Japan), to the cell fluorescence signal of cluster compound dyeing collect (570 ± 20nm), the observation of cell fluorescence imaging.
Result: specifically can be referring to Fig. 1 and 2; The entoblast that from Fig. 1 and 2, can observe through cluster compound mark of the present invention demonstrates very strong fluorescence; Prove that thus labeling method of the present invention can specificity be carried out fluorescence labeling to the living cells entoblast zone of cultivating.
Embodiment 2
Locate fluorescent dye altogether: concrete steps are seen embodiment 1; After the different metal cluster compound dyeing of gold and silver, the PBS washing adds 4% paraformaldehyde immobile liquid 2mL, fixedly 30min; Adding commercialization dyes kernel reagent Syto Green RNA Select (Invitrogen) and further hatches 20min; Sucking-off solution subsequently, PBS washed cell 3 times, each 3～5min.The cell of with 405nm and 488nm laser cluster compound and Syto Green RNA Select being hatched respectively carries out fluorescence signal and collects (the different metal cluster compound of gold and silver: 570 ± 20nm; Syto Green RNA Select:530 ± 20nm)
Result: specifically can be referring to Fig. 3 and 4; From figure, can observe through the entoblast of cluster compound mark of the present invention and can be good at mating, prove that thus labeling method of the present invention can carry out the specificity fluorescent mark to living cells kernel zone with commercialization entoblast staining reagent Syto Green RNA Select.
Embodiment 3
Light stability in cluster compound and the DAPI cell: concrete steps are seen embodiment 1, and after cluster compound dyeing, the PBS washing adds 4% paraformaldehyde immobile liquid 2mL; Fixedly 30min adds organic dyestuff DAPI and further hatches 20min, subsequently sucking-off solution; PBS washed cell 3 times, each 3～5min.Carry out time series scanning through 405nm laser Continuous irradiation cell.
Result: specifically can be referring to Fig. 5 and 6; From figure, can observe and demonstrate very strong fluorescence signal through the entoblast zone of method mark of the present invention; As time goes on (420s); The light stability of cluster compound is high than organic dyestuff DAPI, proves that thus labeling method of the present invention has higher fluorescent stability.
Claims (1)
1. the application of the different metal cluster compound of gold and silver in preparation entoblast fluorescent dye reagent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103408592A (en) * | 2013-08-26 | 2013-11-27 | 厦门大学 | Nine-core gold cluster compound and synthesis method thereof |
| CN104130304A (en) * | 2014-07-07 | 2014-11-05 | 福州大学 | Strong solvent-induced discoloration [Ag6Au6(ethisterone)12] luminous cluster compound and preparation method thereof |
Citations (3)
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|---|---|---|---|---|
| JPH09218202A (en) * | 1995-12-08 | 1997-08-19 | B M L:Kk | Method and kit for detecting anti-nucleus antibody |
| CN101787041A (en) * | 2010-03-10 | 2010-07-28 | 安徽大学 | Copper cluster two-photon absorbing material with living cell developing function and synthetic method thereof |
| SG179378A1 (en) * | 2010-09-17 | 2012-04-27 | Agency Science Tech & Res | Cell-targeting nanoparticles and uses thereof |
-
2012
- 2012-08-06 CN CN2012102772534A patent/CN102798561A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09218202A (en) * | 1995-12-08 | 1997-08-19 | B M L:Kk | Method and kit for detecting anti-nucleus antibody |
| CN101787041A (en) * | 2010-03-10 | 2010-07-28 | 安徽大学 | Copper cluster two-photon absorbing material with living cell developing function and synthetic method thereof |
| SG179378A1 (en) * | 2010-09-17 | 2012-04-27 | Agency Science Tech & Res | Cell-targeting nanoparticles and uses thereof |
Non-Patent Citations (2)
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|---|
| JIAN-HUA JIA,ET AL: "A luminescent gold(I)-copper(I)cluster with unprecedented carbon-centered trigonal prismatic hexagold", 《CHEM.COMMUN》 * |
| JIAN-HUA JIA,ET AL: "Intensely Luminescent Gold(I)-Silver(I)Cluster with Hypercoordinated Carbon", 《J.AM.CHEM.SOC》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103408592A (en) * | 2013-08-26 | 2013-11-27 | 厦门大学 | Nine-core gold cluster compound and synthesis method thereof |
| CN103408592B (en) * | 2013-08-26 | 2015-11-04 | 厦门大学 | A kind of nine core gold duster compound and synthetic methods thereof |
| CN104130304A (en) * | 2014-07-07 | 2014-11-05 | 福州大学 | Strong solvent-induced discoloration [Ag6Au6(ethisterone)12] luminous cluster compound and preparation method thereof |
| CN104130304B (en) * | 2014-07-07 | 2015-12-02 | 福州大学 | A kind of [Ag with strong solvent induced discoloration 6au 6(ethisterone) 12] luminous duster compound and preparation |
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Application publication date: 20121128 |