CN101118236A - Near-infrared fluorescent detecting probe for detecting charged hydrogen in cell, synthetic method and use thereof - Google Patents
Near-infrared fluorescent detecting probe for detecting charged hydrogen in cell, synthetic method and use thereof Download PDFInfo
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- CN101118236A CN101118236A CNA2007100164054A CN200710016405A CN101118236A CN 101118236 A CN101118236 A CN 101118236A CN A2007100164054 A CNA2007100164054 A CN A2007100164054A CN 200710016405 A CN200710016405 A CN 200710016405A CN 101118236 A CN101118236 A CN 101118236A
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- hydrionic
- probe
- hydrogen ion
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- infrared fluorescent
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Abstract
The present invention provides a synthetic process and application as well as a near infrared fluorescent detecting probe for testing intra-cellular hydrogen ion. The process is done by steps as follows: firstly, cyanine dye Cy.7 and hydrogen ion reactive group are dissolved in N, N-dimethylformamide according to the ratio of cyanine dye Cy.7 to hydrogen ion reactive group being one to (one to three), and then, the reaction carries on under 70 to 85 DEG C and with the protection of inert gases for three or five hours; secondly, the reactive compound is cooled to the room temperature and then stirred into diethyl ether anhydrous, the crude product is obtained after filtration, pressure reduction and evaporation; thirdly, the pure product can be extracted with silica gel column chromatography.
Description
Technical field:
The present invention relates to Measurement for Biotechnique and clinical medicine detection range, relate in particular to and be used to detect hydrionic near infrared fluorescent probe in the cell; The invention still further relates to the synthetic method of this fluorescence probe; In addition, this invention also relates to the purposes of this fluorescence probe.
Background technology:
PH is as an important metabolism and an intracellular parameter, in many cell processes, play crucial regulating action, such as cell growth, regulation of calcium, enzymatic activity, signal conduction, endocytosis, chemotactism and other cell processes.Many reports think that some diseases such as cancer, kidney failure, tuberculosis etc. are relevant unusually with the pH in tenuigenin or the acid organelle.Therefore, the accurate measurement of internal pH is very important.
At present, many methods can be used for detecting pH.Such as microelectrode, nuclear-magnetism, absorption and fluorescence spectrum method.In these methods, fluorescence method is owing to the character of its non-invasion, high sensitivity and selectivity, and widely available fluorescent dye is more more superior than additive method when measuring pH.And the fluorescent microscopic imaging technology can capture hydrionic time-space resolution in the cell by using the fluorescent pH probe.
Now, two class fluorescent pH probes are developed, and a class is the probe that is used to detect pH in the tenuigenin, its working pH scope approximately is 6.8-7.4 (K.M.Sun, C.K.McLaughlin, D.R.Lantero and R.A.Manderville, J.Am.Chem.Soc.2007,129,1894-1895; M.S.Briggs, D.D.Burns, M.E.Cooper and S.J.Gregory, Chem.Commun., 2000,2323-2324; J.A.Thomas, R.N.Buchsbaum, A.Zimniak and E.Racker, Biochem.1979,18,2210-2218; A.H.Lee and I.F.Tannock, Cancer Res.1998,58,1901-1908; R.Pal and D.Parker, Chem.Commun.2007,474-476); Another kind of is to be used for detection of acidic organ such as lysosome, the probe of endosome etc., and its working pH scope approximately is 4.5-6.0. (H.-J.Lin, P.Herman, J.S.Kang and J.R.Lakowicz, Anal.Biochem.2001,294,118-125; F.Galindo, M.I.Burguete, L.Vigara, S.V.Luis, N.Kabir, J.Gavrilovic andD.A.Russell, Angew.Chem.Int.Ed.2005,44,6504-6508.).As everyone knows, the subtle change of internal pH may cause the dysfunction of cell, the subtle change of the response internal pH that desirable fluorescence probe should be sensitive, and can avoid interference from cell biological itself.Yet available pH fluorescence probe has following shortcoming at present: low sensitivity, what have excites in the ultraviolet region.Exciting in the ultraviolet region can the damaged tissue sample and cause the interference of biological sample autofluorescence.In addition, it is less to be used for the intraorganic desirable pH probe of detection of acidic, and this is considered to the bottleneck of cell biology and medical development.Therefore, the focus of development pH fluorescence probe is to design near infrared, has good selectivity, high sensitivity, good light stability and working pH be at the fluorescence probe of acid range.
Summary of the invention:
One of purpose of the present invention is intended to overcome the weak point of prior art fluorescence probe, a kind of function admirable is provided, is applicable to and detects hydrionic near infrared fluorescent probe based on the flower cyanine dye in the cell; Two of purpose provides the synthetic method of technology this fluorescence probe simple, with low cost; Three of purpose provides the purposes of this fluorescence probe.
One of purpose of the present invention can realize by following technical measures:
This cyanine dyes fluorescent probe has following general structure
In the general formula: R=-CH
3,-CH
2CH
3,-CH
2CH
2CH
3,-CH
2CH
2CH
2CH
2SO
3 -
-OH is positioned at amino ortho position, a position or contraposition.
Two of purpose of the present invention can realize by following technical measures:
This method is carried out as follows:
A. earlier with cyanine dye Cy.7 and hydrogen ion response group according to cyanine dye Cy.7: the mass ratio of hydrogen ion response group=1: 1～3 is dissolved among the DMF (N, dinethylformamide), then in inert gas shielding and 70-85 ℃ reaction 3～5 hours down;
B. reaction mixture is cooled to room temperature, adds in the absolute ether in stirring down, after filtration, drying under reduced pressure gets crude product;
C. silica gel column chromatography separate pure product.
Two of purpose of the present invention also can realize by following technical measures:
Colored cyanine type dye Cy.7 described in a operation is selected from methyl flower cyanines, ethyl flower cyanines, propyl group flower cyanines or sulfonic group butyl flower cyanines; Hydrogen ion response group described in a operation is selected from m-aminophenol, o-aminophenol or para-aminophenol; Absolute ether described in the b operation: reaction mixture=15～20: 1 volume ratio.
In the representational compound exhibits of the present invention synthetic schemes below.
Under inert gas shielding, the reaction in DMF (N, dinethylformamide) of flower cyanine dye and m-aminophenol obtains probe molecule, at last through separating the pure product of probe that obtain.
The present invention detects the interior hydrionic near infrared fluorescent probe of cell does not have special restriction in use, usually, probe molecule can be dissolved in physiological saline, damping fluid or, add in the suitable damping fluid that contains cell tissue then and test by water-miscible organic solvents such as ethanol, acetonitrile, dimethyl sulfoxides.
Three of purpose of the present invention can realize by following technical measures:
Hydrionic near infrared fluorescent probe is used for the hydrionic detection of chemical simulation living things system in the detection cell of the present invention, hydrionic analyzing and testing and fluorescence imaging in biological living cells and the living tissue detect, and hydrionic detection in the pathological tissues on the clinical medicine.
The present invention is a raw material with propyl group flower cyanines, m-aminophenol, synthetic fluorescence probe
1H NMR (300MHz, CDCl
3): δ 1.04 (t, 6H, J=7.5Hz), 1.27 (s, 12H), 1.83 (m, 2H), 2.06 (m, 4H), 2.58 (m, 4H), 3.89 (t, 4H, J=6.8Hz), 5.84 (d, 2H, J=14.0Hz), 6.45 (m, 4H), 6.92-7.23 (m, 8H), 8.08 (d, 2H, J=14.0Hz) .ESI-MS cal for[M]
+=612.4, found 612.4 (M-I
-).
Significant contribution of the present invention is that the fluorescent probe molecule that synthesizes belongs near infrared fluorescent probe, can effectively avoid the interference of autofluorescence in the cell, improves the selectivity and the sensitivity of detection method, reduces the damage to life entity, helps live body and detects; The design of fluorescent probe molecule of the present invention is based on inducing electron transfer (PET) in the molecule, this probe has shown high sensitivity, different excite with emission wavelength under can make response respectively to the pH under two kinds of conditions of soda acid.The pH titration experiments shows that can increase by 10 times in the pH of 4.0-6.5 scope fluorescence intensity under the acid condition has a surplus, and pKa is 5.14, and this is very significant to studying intracellular acid organ; Under alkali condition, the working pH scope is 10.5-11.8, and pKa is 11.31.In addition, probe is applied to (HepG2cells) in people's the hepatoma carcinoma cell further proved this probe by the laser co-focusing micro-imaging technique value.
The probe molecule that the present invention relates to has extremely important using value.Particularly should be positioned at the near-infrared region by series probe molecule emission wavelength, and can realize the double check of pH, and measure highly sensitively, selectivity is good, and the perviousness and the toxic and side effect of pair cell are little.In addition should the series probe molecule simple in structure, high the making of synthetic simple and easy and efficient be very easily promoted practical application.
Description of drawings:
Fig. 1 is the relation and the pH titration curve of the fluorescent probe molecule of the embodiment of the invention its fluorescence intensity and pH in acidic buffer solution.Horizontal ordinate is wavelength (nm), and ordinate is a fluorescence intensity.
Fig. 2 is the relation and the pH titration curve of the fluorescent probe molecule of the embodiment of the invention its fluorescence intensity and pH in alkaline buffer solution.Horizontal ordinate is wavelength (nm), and ordinate is a fluorescence intensity.
Fig. 3 is that the fluorescent probe molecule of the embodiment of the invention has good selectivity to hydrogen ion, and horizontal ordinate is various ions, 1:H
+2:H
++ Cu
2+3:H
++ Zn
2+4:H
++ Ca
2+5:H
++ Mg
2+Ordinate is for adding the fluorescence intensity of various ions front and back probe solution.
Fig. 4 is the laser co-focusing micro-imaging of the fluorescent probe molecule of the embodiment of the invention to people's hepatoma carcinoma cell (HepG2cells).A: people's hepatoma carcinoma cell is hatched 1 hour laser co-focusing photo under 37 ℃ with fluorescence probe; B: the bright field photo is to show cell viability.
Embodiment:
Embodiment 1:
Synthesizing of probe
Get propyl group cyanine dye Cy.7,5.2 gram m-aminophenol and 40mL N that 1.4 grams (2mmol) have near-infrared fluorescent spectrum; dinethylformamide is in the 100mL there-necked flask; it is back nitrogen protection and 70 ℃ of following reactions 3 hours that it is dissolved fully; be cooled to room temperature; the back joins in the 940mL ether under stirring; filter; drying under reduced pressure gets the fluorescence probe crude product; use the silicagel column separation component at last; eluant, eluent is selected from methyl alcohol-ethyl acetate (1: 20v/v); 35 ℃ of rotary evaporations are removed solvent, and vacuum drying gets the pure product of fluorescence probe.
Embodiment 2:
Synthesizing of probe
Get propyl group cyanine dye Cy.7,1.4 gram m-aminophenol and 20mL N that 1.4 grams (2mmol) have near-infrared fluorescent spectrum; dinethylformamide is in the 100mL there-necked flask; it is back nitrogen protection and 85 ℃ of following reactions 5 hours that it is dissolved fully; be cooled to room temperature; the back joins in the 350mL ether under stirring; filter; drying under reduced pressure gets the fluorescence probe crude product; use the silicagel column separation component at last; eluant, eluent is selected from methyl alcohol-ethyl acetate (1: 20v/v); 38 ℃ of rotary evaporations are removed solvent, and vacuum drying gets the pure product of fluorescence probe.
Embodiment 3:
Synthesizing of probe
Get propyl group cyanine dye Cy.7,2.5 gram m-aminophenol and 28mL N that 1.4 grams (2mmol) have near-infrared fluorescent spectrum; dinethylformamide is in the 100mL there-necked flask; it is back nitrogen protection and 75 ℃ of following reactions 4 hours that it is dissolved fully; be cooled to room temperature; the back joins in the 550mL ether under stirring; filter; drying under reduced pressure gets the fluorescence probe crude product; use the silicagel column separation component at last; eluant, eluent is selected from methyl alcohol-ethyl acetate (1: 20v/v); 30 ℃ of rotary evaporations are removed solvent, and vacuum drying gets the pure product of fluorescence probe.
Embodiment 4:
Methyl flower cyanines are replaced propyl group flower cyanines, and other are respectively with embodiment 1,2,3.
Embodiment 5:
Ethyl flower cyanines are replaced propyl group flower cyanines, and other are respectively with embodiment 1,2,3.
Embodiment 6:
Sulfonic group butyl flower cyanines are replaced propyl group flower cyanines, and other are respectively with embodiment 1,2,3.
Embodiment 7:
O-aminophenol is replaced m-aminophenol, and other are respectively with embodiment 1,2,3,4,5,6.
Embodiment 8:
Para-aminophenol is replaced m-aminophenol, and other are respectively with embodiment 1,2,3,4,5,6.
Claims (6)
1. hydrionic near infrared fluorescent probe in the detection cell is characterized in that this fluorescence probe has following general structure:
In the general formula: R=-CH
3,-CH
2CH
3,-CH
2CH
2CH
3,-CH
2CH
2CH
2CH
2SO
3 -
-OH is positioned at amino ortho position, a position or contraposition.
2. the synthetic method of hydrionic near infrared fluorescent probe in the detection cell is characterized in that this method carries out as follows:
A. earlier with cyanine dye Cy.7 and hydrogen ion response group according to cyanine dye Cy.7: the mass ratio of hydrogen ion response group=1: 1～3 is dissolved in N, in the dinethylformamide, then in inert gas shielding and 70-85 ℃ reaction 3～5 hours down;
B. reaction mixture is cooled to room temperature, adds in the absolute ether in stirring down, after filtration, drying under reduced pressure gets crude product;
C. silica gel column chromatography separate pure product.
3. synthetic method according to claim 2 is characterized in that the colored cyanine type dye Cy.7 described in a operation is selected from methyl flower cyanines, ethyl flower cyanines, propyl group flower cyanines or sulfonic group butyl flower cyanines.
4. synthetic method according to claim 2 is characterized in that the hydrogen ion response group described in a operation is selected from m-aminophenol, o-aminophenol or para-aminophenol.
5. synthetic method according to claim 2 is characterized in that the absolute ether described in the b operation: reaction mixture=15～20: 1 volume ratio.
6. hydrionic near infrared fluorescent probe is used for the hydrionic detection of chemical simulation living things system in the described detection cell of claim 1, hydrionic analyzing and testing and fluorescence imaging in biological living cells and the living tissue detect, and hydrionic detection in the pathological tissues on the clinical medicine.
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|---|---|---|---|
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507519A (en) * | 2011-10-26 | 2012-06-20 | 华南师范大学 | Application of dansyl acid serving as pH fluorescent probe |
| CN101811870B (en) * | 2009-07-21 | 2012-09-05 | 哈尔滨理工大学 | Probe for quick tester of hydrogen content in aluminium alloy melt and manufacturing method thereof |
| CN103555317A (en) * | 2013-10-08 | 2014-02-05 | 东南大学 | PH sensitive near-infrared fluorescence molecular probe and preparation method and use thereof |
| CN104673274A (en) * | 2013-12-02 | 2015-06-03 | 复旦大学 | Acid-sensing near-infrared fluorescence probe for targeting tracing of tumor metastasis tendency evaluation |
| CN105153018A (en) * | 2014-12-18 | 2015-12-16 | 北京工商大学 | Hemicyanine derivative pH fluorescence ratio sensor |
| CN109770853A (en) * | 2019-01-30 | 2019-05-21 | 上海师范大学 | A kind of zebra fish wound detection method |
| CN109776379A (en) * | 2019-03-08 | 2019-05-21 | 武汉大学 | It is a kind of to can be used for responding the near infrared fluorescent probe and preparation method thereof that in living cells and pH changes in chronic wounds development process |
| CN110551056A (en) * | 2019-09-29 | 2019-12-10 | 山东师范大学 | Cyanine compound, preparation method and application in detection of Golgi pH |
| CN111269287A (en) * | 2020-02-06 | 2020-06-12 | 苏州大学 | Activatable optical molecular probe and preparation method and application thereof |
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2007
- 2007-07-24 CN CNA2007100164054A patent/CN101118236A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101811870B (en) * | 2009-07-21 | 2012-09-05 | 哈尔滨理工大学 | Probe for quick tester of hydrogen content in aluminium alloy melt and manufacturing method thereof |
| CN102507519A (en) * | 2011-10-26 | 2012-06-20 | 华南师范大学 | Application of dansyl acid serving as pH fluorescent probe |
| CN102507519B (en) * | 2011-10-26 | 2013-08-28 | 华南师范大学 | Application of dansyl acid serving as pH fluorescent probe |
| CN103555317A (en) * | 2013-10-08 | 2014-02-05 | 东南大学 | PH sensitive near-infrared fluorescence molecular probe and preparation method and use thereof |
| CN104673274A (en) * | 2013-12-02 | 2015-06-03 | 复旦大学 | Acid-sensing near-infrared fluorescence probe for targeting tracing of tumor metastasis tendency evaluation |
| CN105153018B (en) * | 2014-12-18 | 2017-09-22 | 北京工商大学 | A kind of half cyanines derivative pH ratio fluorescent sensors |
| CN105153018A (en) * | 2014-12-18 | 2015-12-16 | 北京工商大学 | Hemicyanine derivative pH fluorescence ratio sensor |
| CN109770853A (en) * | 2019-01-30 | 2019-05-21 | 上海师范大学 | A kind of zebra fish wound detection method |
| CN109770853B (en) * | 2019-01-30 | 2022-01-14 | 上海师范大学 | Zebra fish wound detection method |
| CN109776379A (en) * | 2019-03-08 | 2019-05-21 | 武汉大学 | It is a kind of to can be used for responding the near infrared fluorescent probe and preparation method thereof that in living cells and pH changes in chronic wounds development process |
| CN109776379B (en) * | 2019-03-08 | 2021-01-15 | 武汉大学 | Near-infrared fluorescent probe capable of responding to pH change in living cells and in chronic wound development process and preparation method thereof |
| CN110551056A (en) * | 2019-09-29 | 2019-12-10 | 山东师范大学 | Cyanine compound, preparation method and application in detection of Golgi pH |
| CN111269287A (en) * | 2020-02-06 | 2020-06-12 | 苏州大学 | Activatable optical molecular probe and preparation method and application thereof |
| CN111269287B (en) * | 2020-02-06 | 2022-02-15 | 苏州大学 | Activatable optical molecular probe and preparation method and application thereof |
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Open date: 20080206 |