CN102676154A - Application of phosphorus phthalocyanine and phosphorus phthalocyanine derivative serving as fluorescence probes - Google Patents
Application of phosphorus phthalocyanine and phosphorus phthalocyanine derivative serving as fluorescence probes Download PDFInfo
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- CN102676154A CN102676154A CN2011100595013A CN201110059501A CN102676154A CN 102676154 A CN102676154 A CN 102676154A CN 2011100595013 A CN2011100595013 A CN 2011100595013A CN 201110059501 A CN201110059501 A CN 201110059501A CN 102676154 A CN102676154 A CN 102676154A
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- phosphorus phthalocyanine
- phthalocyanine
- tbc
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Abstract
The invention discloses application of phosphorus phthalocyanine and phosphorus phthalocyanine derivative serving as fluorescence probes. Fluorescence imaging effects show that the phosphorus phthalocyanine and the derivative of the phosphorus phthalocyanine have characteristic fluorescence peaks and the fluorescence peaks can avoid interference of autofluorescence in any situations. Compared with [P(OH)2TBC], P(OH)2TBC (Beta-OiPr) 4 can penetrates through a cell membrane and is enriched on a mitochondria portion, and the labeling efficiency of the P(OH)2TBC (Beta-OiPr) 4 is obviously higher than the transfection fluorescence labeling efficiency. The phosphorus phthalocyanine and the derivative of the phosphorus phthalocyanine are good fluorescence labeling probes and can be positioned on mitochondria inside cells and perform fluorescence labeling of the mitochondria.
Description
Technical field
The present invention relates to the application of Phosphorus phthalocyanine and verivate thereof as fluorescent probe.
Background technology
Along with the completion of the Human Genome Project, the albumen of pair cell subprovince, substructure is formed and the emphasis that dynamic analysis becomes research is carried out in interaction.Utilizing fluorescent mark to carry out bio-imaging is the important means of carrying out this research.Fluorescent probe technique plays an important role in bio-imaging, can carry out the effect of spike to research object.
Because the detection sensitivity of fluorescence imaging largely depends on the luminous intensity and the photochemical stability of probe, the core realm of therefore having studied for fluorescence imaging to the research and development of probe.Present widely used bio-imaging is used fluorescent probe, mainly contains nano luminescent materials such as small molecules luminescent material, GFP and semiconductor-quantum-point, carbon nanotube such as organic fluorescent dye by the chemical group composition.Wherein, the most widely used is organic fluorescent dye, semiconductor-quantum-point (QDs), GFP (Chalfie M, Tu Y; Euskirchen G, Ward WW, Prasher DC.Greenfluorescent protein as a marker for gene expression.Science; 1994,263 (5148): 802-805.Glepmans BNG, Adams SR; Ellisman MH; Tsien RY.The fluorescent toolbox for assessing protein location and function.Science, 2006,312 (5771): 217-224.).
In modern times in the biology, viable cell unit molecule behavioral study, and the field, forward position of living animal imaging having become biological imaging.No matter be living animal and viable cell, the autofluorescence of biological sample is very serious to the interference of tagged molecule signal.Therefore, the exploitation good light stability, toxicity is low and have the luminescent material of specific fluorescence characteristic to become the fresh target of bio-imaging probe research.
Summary of the invention
The purpose of this invention is to provide the application of Phosphorus phthalocyanine and verivate thereof as fluorescent probe.
Phosphorus phthalocyanine compound according to the invention is that phosphorus four benzos three azepines are breathed out sieve [P (OH)
2TBC], structural formula is seen formula I; Its verivate is the Phosphorus phthalocyanine of tetraisopropoxide [P (OH)
2TBC (β-O
iPr)
4], structural formula is seen formula II.Above-claimed cpd all can become to obtain through big cyclization.
(formula I) (formula II)
Phosphorus phthalocyanine that the present invention relates to and verivate thereof have biocompatible amphipathic, reach light stability preferably, have the basis that becomes fluorescent probe.They have two photoluminescent properties in viable cell, may be used on the bio-imaging field, are used for Intramitochondrial fluorescence location as fluorescent probe.
Phosphorus phthalocyanine and derivatives fluorescent imaging effect thereof are shown that Phosphorus phthalocyanine and verivate thereof have the very fluorescence peak of characteristic, can under any circumstance avoid the interference of autofluorescence.Through with [P (OH)
2TBC] comparison, P (OH)
2TBC (β-O
iPr)
4More be prone to permeates cell membranes and be enriched in the plastosome position, P (OH)
2TBC (β-O
iPr)
4Labeling effciency apparently higher than the fluorescent label efficiency of transfection.Phosphorus phthalocyanine and verivate thereof are the fluorescence labeling probes of one type of excellence, will play a significant role aspect relevant viable cell imaging such as plastosome fluorescence labeling probe and the research.
Description of drawings
Fig. 1 is P (OH)
2TBC adds fluorescent microscope photo behind the Hela cell cultures 12h with 10 μ g/ml; Wherein (a) is the imaging of UV excited fluorescent; (b) be blue-light excited fluorescence imaging; (c) be the imaging of green glow excited fluorescent; (d) light field imaging.
Fig. 2 is P (OH)
2TBC uses 405nm laser excitation after adding Hela cell cultures 12h with 10 μ g/ml, the fluorescence imaging in the scanning 423nm-721nm scope.
Fig. 3 is P (OH)
2TBC uses 488nm laser excitation after adding Hela cell cultures 12h with 10 μ g/ml, the fluorescence imaging in the scanning 500nm-721nm scope.
Fig. 4 is P (OH)
2TBC uses 543nm laser excitation after adding Hela cell cultures 12h with 10 μ g/ml, the fluorescence imaging in the scanning 558nm-721nm scope.
Fig. 5 is plastosome mark EGFP (enhanced green fluorescence protein), P (OH)
2TBC and verivate thereof (P (OH)
2TBC (β-O
iPr)
4) with behind the 10 μ g/ml adding Hela cell cultures 12h, excite with 488nm, gather the EGFP image, 543nm laser is gathered P (OH) 2TBC and verivate (P (OH) thereof
2TBC (β-O
iPr)
4) stripped fluoroscopic image; (a) the Hela cell does not add photosensitizers; (b) add P (OH)
2TBC; (c) add P (OH)
2TBC (β-O
iPr)
4
Embodiment
Through specific embodiment purposes of the present invention is further specified below.
Experimental technique described in the following embodiment like no specified otherwise, is ordinary method; Said reagent and biomaterial like no specified otherwise, all can obtain from commercial sources.
Used P (OH) among the embodiment
2TBC and P (OH)
2TBC (β-O
iPr)
4Be to prepare: L.Huang with reference to the synthetic route in the following document; P.Zhao; F.Zhang; Z.Li, C.-H.Tung.Synthesis and DualFluorescence Property of Novel Dihydroxy Phosphorus TetrabenzotriazacorroleDerivates.Proc.SPIE.2007,682714..Synthetic compound confirms that through mass spectrum, ultimate analysis, nuclear-magnetism its structure is a target compound.
The viable cell fluorescence imaging of embodiment 1, photosensitizers is observed
(in vitro) studies fat-soluble P (OH) in the cell in vitro level
2TBC (β-O
iPr)
4And P (OH)
2TBC comprises that to human cervical carcinoma Hela cell's imaging effect photosensitizers adds the fluorescence imaging of back Hela cell and the Subcellular Localization situation of photosensitizers.
With synthetic photosensitizers (P (OH)
2TBC or P (OH)
2TBC (β-O
iPr)
4) join in the HeLa cell, lucifuge was cultivated 12 hours, used Olympus IX71 (Japan) inverted fluorescence microscope, excited with the X-Cite metal halide lamp, observed respectively through the UV/BLU/GRE filter.Use the 20X object lens of object lens as LUCPlanFLN, N.A. is 0.45.The result is as shown in Figure 1, and wherein (a) is the imaging of UV excited fluorescent; (b) be blue-light excited fluorescence imaging; (c) be the imaging of green glow excited fluorescent; (d) light field imaging.
Under the fluorescent microscope, observe photosensitizers and be absorbed in the cell, and send blueness respectively, green and red fluorescence, like Fig. 1.Show according to the fluorescence spectrum of Phosphorus phthalocyanine in solution, when photosensitizers can go out two fluorescence peaks on the fluorescence spectrum during by short wavelength's optical excitation.Supposition is the fluorescence in blue-greenish colour zone under intracellular environment near the fluorescence peak of shortwave direction wherein.
The laser co-focusing spectral investigation of embodiment 2, photosensitizer fluorescence imaging
Photosensitizers viable cell fluorescence imaging situation is further observed through Laser Scanning Confocal Microscope, and the laser confocal microscope model of using is Zeiss LSM710 (Germany).Adopt the micro-viable cell culture systems of Chamlide TC of Korea S LCI company, keeping the Hela cell is 5% CO at 37 ℃, volume(tric)fraction
2, observe under the saturated humidity condition.
Because Phosphorus phthalocyanine (P (OH)
2TBC) and verivate (P (OH)
2TBC (β-O
iPr)
4) unknown at intracellular fluorescence spectrum, therefore fixedly excitation wavelength adopts full spectral scan method to detect.Add Phosphorus phthalocyanine lucifuge and cultivate 12h, use solid statelaser 405nm laser to be exciting light, under laser confocal microscope, carry out λ scanning, the fluorescence imaging effect that obtains, as shown in Figure 2.Can find out that when exciting light is 405nm Phosphorus phthalocyanine is 442nm-548nm at the intracellular fluorescence spectra of Hela.
When exciting light is 488nm, as shown in Figure 3, under laser confocal microscope, carry out λ scanning, obtaining Phosphorus phthalocyanine is 615nm-673nm at the intracellular fluorescence spectra of Hela.When exciting light is the 543nm light time, the 558nm-721nm scope is detected, obtaining Phosphorus phthalocyanine is 558nm-634nm in the intracellular fluorescence spectra scope of Hela, like Fig. 4.When exciting light changed, the fluorescent emission scope also can change to some extent, but is that the Phosphorus phthalocyanine that 488nm or the optical excitation of 543nm go out all belongs to the fluorescence in long wavelength's red range at the intracellular fluorescence of Hela, was the corresponding S1 attitude fluorescence of Phosphorus phthalocyanine.And only under 405nm or shorter wavelength, excite be only S2 attitude fluorescence, being absorbed the back by the Hela cell is blue-greenish colour fluorescence.
Embodiment 3, the photosensitizers plastosome location in viable cell
After fluorescence emission spectrum after Phosphorus phthalocyanine is absorbed by the Hela cell is confirmed, further its location is observed.Add Phosphorus phthalocyanine lucifuge and cultivate that the fluorescence imaging at Hela endocellular phosphorus class phthalocyanine is wire behind the 12h, also have granular or the short-term shape, diameter is generally at 0.5～1.0 μ m.Infer for plastosome and locate, and then adopt the plastosome of electric commentaries on classics method mark Hela cell, employing be labeled as EGFP.Select two channel scanning methods during observation, copolymerization Jiao goes up the dye range of selecting EGFP and Rhodamin, and promptly 488nm excites, and 492nm-525nm gathers the image of EGFP; 543nm excites 565nm-685nm to gather the fluoroscopic image of Phosphorus phthalocyanine and derivative photosensitizer (PS) thereof, and as shown in Figure 5, wherein (a) Hela cell does not add photosensitizers; (b) the Hela cell adds P (OH)
2TBC; (c) the Hela cell adds P (OH)
2TBC (β-O
iPr)
4
At intracellular fluorescence, can find out that both have significantly location altogether through alternative line plastochondria fluorescence and Phosphorus phthalocyanine and verivate thereof.At P (OH)
2TBC (β-O
iPr)
4Image in the fluorescent label efficiency of transfection be starkly lower than the labeling effciency of Phosphorus phthalocyanine derivates, the Hela cell is redfree fluorescence when not adding photosensitizers, cell all has been labeled the plastosome red fluorescence when adding photosensitizers.P (OH)
2TBC (β-O
iPr)
4More be prone to permeates cell membranes and be enriched in the plastosome position, the labeling effciency of Phosphorus phthalocyanine derivates is apparently higher than the fluorescent label efficiency of transfection.It is the fluorescence labeling probe of one type of excellence.
Claims (4)
1. four benzos of the phosphorus shown in the formula I, three azepines are breathed out the application of the Phosphorus phthalocyanine of tetraisopropoxide shown in sieve and/or the formula II as fluorescent probe;
(formula I) (formula II).
2. application according to claim 1 is characterized in that: said fluorescent probe is used for the intracellular plastosome of telltale mark.
3. four benzos of the phosphorus shown in the formula I, three azepines are breathed out the application of the Phosphorus phthalocyanine of tetraisopropoxide shown in sieve and/or the formula II in the preparation fluorescent probe.
4. application according to claim 3 is characterized in that: said fluorescent probe is used for the intracellular plastosome of telltale mark.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106547168A (en) * | 2016-10-28 | 2017-03-29 | 深圳市华星光电技术有限公司 | A kind of black-matrix material compositionss and application |
| CN109283161A (en) * | 2018-09-19 | 2019-01-29 | 青岛大学 | A kind of method using peroxide value in fluorescent quenching method detection grease |
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2011
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Patent Citations (4)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106547168A (en) * | 2016-10-28 | 2017-03-29 | 深圳市华星光电技术有限公司 | A kind of black-matrix material compositionss and application |
| CN109283161A (en) * | 2018-09-19 | 2019-01-29 | 青岛大学 | A kind of method using peroxide value in fluorescent quenching method detection grease |
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| CN102676154B (en) | 2014-01-29 |
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