CN101671555A - Nitric oxide fluorescent probe based on ruthenium (II) complex and application thereof - Google Patents

Nitric oxide fluorescent probe based on ruthenium (II) complex and application thereof Download PDF

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CN101671555A
CN101671555A CN200910308259A CN200910308259A CN101671555A CN 101671555 A CN101671555 A CN 101671555A CN 200910308259 A CN200910308259 A CN 200910308259A CN 200910308259 A CN200910308259 A CN 200910308259A CN 101671555 A CN101671555 A CN 101671555A
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bpy
ruthenium
nitric oxide
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袁景利
张润
叶志强
王桂兰
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Dalian University of Technology
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Abstract

The invention relates to a novel ruthenium (II) complex fluorescent probe used for measuring nitric oxide imaging in living cells and the application thereof. The probe is the complex formed by ruthenium (II), 2 -bipyridine and derivants thereof. The structure of the complex is a right formula; when the complex and the living cells are cultivated together, the complex can enter the living cells, and further specially captures nitric oxide in the cells, leads the fluorescence intensity of the complex to be obviously enhanced; and then is used for the fluorescent imaging measurement of the nitric oxide in the living cells.

Description

A kind of nitric oxide fluorescent probe and application thereof based on ruthenium (II) title complex
Technical field
The present invention relates to the determination techniques field of viable cell intracellular nitric oxide, specifically a kind of ruthenium (II) coordination compound fluorescent probe and application thereof that can be used for viable cell intracellular nitric oxide fluorescence imaging mensuration.
Background technology
Nitrogen protoxide (NO) is a kind of highly active paramagnetism free radical molecule, on its antibonding p track a unpaired electron is arranged, and is widespread in nature.It is can be by organism automatically synthetic and can be in animal, plant, fungi, bacterial body and some other free radical molecule or metalloprotein rapid reaction, thus performance important physical, pathological effect.In vivo, the NO of lower concentration is as in the cell and a kind of signal transduction molecule of iuntercellular play an important role in blood circulation, immunity, neural system (document 1:R.M.J.Palmer, A.G.Ferrige, S.Moncada, Nature1987,327,524; Document 2:A.R.Butler, D.L.H.Williams, Chem.Soc.Rev.1993,22,233; Document 3:R.F.Furchgott, Angew.Chem.Int.Ed.1999,38,1870; Document 4:L.J.Ignarro, Angew.Chem.Int.Ed.1999,38,1882), and when the excessive concentration of NO, just with body in other active oxygen species (ROS) reaction produce a large amount of active nitrogen species (RNS) (document 5:F.L.M.Ricciardolo, P.J.Sterk, B.Gaston, G.Folkerts, Physiol.Rev.2004,84,731), and then to biomolecules such as nucleic acid, fat and protein cause damage.
Because NO has very important physiological action, the NO detection method of setting up in sensitive the NO detection method, particularly living things system more and more causes people's attention.At present, NO detects and mainly contains following several method:
(1) utilizing NO is a kind of magnetic resonance spectroscopy (document 6:K.Sancier, G.Freeman, I.Mills, Science1962,137,752 of free radical feature; Document 7:T.Nagano, T.Yoshimura, Chem.Rev.2002,102,1235), still, because NO transformation period in liquid phase is extremely short, as easy as rolling off a log other oxynitride that is oxidized to makes on this method sensitivity, specificity, stability and the range of application slightly not enough.
(2) spectrophotometry utilizes NO easily to be oxidized to NO 2 -And then under acidic conditions, generate method (document 8:L.C.Green, D.A.Wagner, the J.Glogowski of diazotization thing with the Griess reagent react, P.L.Skipper, J.S.Wishnok, S.R.Tannenbaum, Anal.Biochem.1982,126,131), this method is simple to operate, but accuracy, sensitivity are lower.
(3) electrochemical process utilizes NO easily oxidized and electrochemical reaction takes place, thus to NO concentration carry out method for measuring (Nature 1992,358 for document 9:T.Malinski, Z.Taha, 676; Document 10:F.Bedioui, N.Villeneuve, Electroanal.2003,15,15), this method is highly sensitive, electrode stability good, long service life, but can be used for the electrode making difficulty that the interior NO of cell measures, and kind electrode is inserted the bigger damage of cell meeting pair cell generation.
(4) chemoluminescence method, with oxygenant NO is oxidized to and releases energy and luminous method (document 11:J.F.Brien, B.E.McLaughlin, K.Nakatsu when utilizing excited state nitrogen peroxide to return ground state behind the nitrogen peroxide, G.S.Marks, MethodsEnzymol.1996,268,83), this method is highly sensitive, but complicated operation, and the infringement of oxygenant that uses in operating process such as pair cells such as ozone, hydrogen peroxide is very big, is difficult to the mensuration of NO in the cell system.
(5) fluorometry (document 12:H.Kojima, N.Nakatsubo, K.Kikuchi, S.Kawahara, Y.Kirino, H.Nagoshi, Y.Hirata, T.Nagano, Anal.Chem.1998,70,2446 of use specific probe; Document 13:M.H.Lim, D.Xu, S.J.Lippard, Nat.Chem.Biol.2006,2,375; Document 14:E.W.Miller, C.J.Chang, Curr.Opin.Chem.Biol.2007,11,620), characteristics such as that this method has is simple to operate, highly sensitive, selectivity is good are one of at present ideal detection methods.But the employed fluorophore of present existing NO fluorescent probe all is the organic fluorescence molecule, these organic fluorescence groups exist light stability poor, easily by photobleaching, the pH use range is narrower, the Stokes displacement is less, easily when measuring because of exciting shortcoming (document 15:Z.Zhang such as the fluorescence self-quenching takes place with scattering, S.Achilefu, Org.Lett.2004,6,2067).In addition, though these fluorescent probe molecules can pass cytolemma and intracellular NO reacts and the generation fluorescent signal, the reaction product of these probe molecules and NO also is easy to overflow from cell, thereby produces error when fluorometric assay.
Summary of the invention
The technical problem to be solved in the present invention provide a kind of highly sensitive, selectivity and good water solubility, applied widely, can be used for the NO imaging is measured in the viable cell ruthenium (II) coordination compound fluorescent probe and application.
Technical scheme of the present invention is as follows:
With bipyridine and derivative thereof is the title complex that part and divalent ruthenium form, and two (2,2 '-bipyridine) (4-(3,4 diamino phenoxy)-2,2 '-bipyridine) closes ruthenium (II) (hereinafter to be referred as Ru (bpy) 2 (dabpy) 2+), being fluorescent probe, wherein said fluorescent probe structural formula is:
Figure A20091030825900041
Ru(bpy) 2(dabpy) 2+
The application process of described nitric oxide fluorescent probe based on ruthenium (II) title complex is: in all kinds of biologies and abiotic environment, utilize described ruthenium (II) coordination compound fluorescent probe to catch NO in the system, make the fluorescence intensity of probe significantly strengthen, determine generation and the growing amount of NO then by fluorometry.Described fluorometry comprises conventional fluorometry or fluorescent microscope imaging assay method.
Described determination of nitric oxide can be used for containing in the mensuration test kit and related reagent of its component with ruthenium (II) coordination compound fluorescent probe.
Fluorescent probe of the present invention has following advantage:
1. have well water-solublely, be applicable to nitric oxide production mensuration in the living things systems such as cell and biological tissue.
2. stability is high, can prolonged preservation use, and is applicable to multiple environment such as slightly acidic, neutrality and alkalescence.
3. have higher NO and measure sensitivity, be limited to 0.27mmol/L under the lowest detection.
4. NO there is good selectivity, makes almost no change of time spent fluorescent signal with other active oxygen species.
5. under oxygen coexistence, can cause the fluorescence intensity enhancing with the NO reaction rapidly, 16.9 times of its fluorescence quantum efficiency increases.
6. under common culture condition, can enter in the viable cell, with intracellular nitrogen protoxide reaction, cause that the fluorescence intensity of title complex significantly strengthens, and then be used for the nitric oxide production fluorometric assay of viable cell specifically.
7. the product of the reaction of probe and NO is (hereinafter to be referred as Ru (bpy) 2(T-bpy) 2+) can not pass cytolemma and be diffused into outside the film, thereby help the accurate fluorometric assay of NO in the cell more.
Description of drawings
Fig. 1 is Ru (bpy) 2(dabpy) 2+And Ru (bpy) 2(T-bpy) 2+Synthetic route chart.
Fig. 2 is Ru (bpy) 2(dabpy) 2+(solid line, 10mmol/L) and Ru (bpy) 2(T-bpy) 2+(dotted line, 10mmol/L) the fluorescence spectrum figure in the 0.1mol/L of pH value 7.4 phosphate buffer solution.
Fig. 3 is Ru (bpy) 2(dabpy) 2+(B, 10mmol/L) and Ru (bpy) 2(T-bpy) 2+(A, 10mmol/L) the fluorescence intensity variation diagram in the 0.1mol/L of different pH values phosphate buffer solution.
Fig. 4 is Ru (bpy) 2(dabpy) 2+In the 0.1mol/L of pH value 7.4 borate buffer solution, relatively scheme with the fluorescence intensity of various active oxygen species reaction product.
Fig. 5 is Ru (bpy) 2(dabpy) 2+In the 0.1mol/L of pH value 7.4 borate buffer solution, react the changing conditions figure of its product fluorescence spectrum afterwards with different amount NO saturated solutions.
Fig. 6 is Ru (bpy) 2(dabpy) 2+In the 0.1mol/L of pH value 7.4 borate buffer solution, detect the working curve diagram of NO.
Fig. 7 is Ru (bpy) 2(dabpy) 2+The mouse macrophage of mark is at no NO and the light field imaging and the fluorescence imaging measurement result figure that have in the presence of the NO.
Fig. 8 is Ru (bpy) 2(dabpy) 2+Light field imaging and the fluorescence imaging measurement result figure of the cape jasmine cell of mark under different incubation times.
Fig. 9 is Ru (bpy) 2(T-bpy) 2+Cultivate the light field imaging and the fluorescence imaging measurement result figure of cell after 5 hours with the cape jasmine cell.
Embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme and accompanying drawing.Present embodiment only is used for that the present invention will be described, also belongs to scope of the present invention based on the method for same principle and similar raw material.
Embodiment 1: title complex [Ru (bpy) 2(dabpy)] (PF 6) 2[Ru (bpy) 2(T-bpy) 2+] (PF 6) 2Synthetic
Synthetic route as shown in Figure 1, the matrix operating process is as follows.
(1) two (2,2 '-bipyridine) (4-chloro-2,2 '-bipyridine) closes the synthetic of ruthenium (II) (compound 1)
To contain 38.1 milligrams of 4-chloro-2,2 '-bipyridine (0.2mmol) and 104.1 milligrams suitable-two (2,2 '-bipyridine)-two chloro-two hydration rutheniums (II) 60mL methanol solution reflux (0.2mmol) evaporate to dryness after 6 hours, thick product separates with silica gel column chromatography, with 100: 7: CH O.5 3CN-H 2O-KNO 3Solution is eluent.Decompression steams a spot of CH of product behind the solvent 3CN-H 2O dissolving back adds saturated phosphofluoric acid aqueous ammonium separates out title complex.Filter collecting precipitation, and, get 126.9 milligrams of target compounds, productive rate: 71% after the vacuum-drying with the less water washing. 1HNMR (CD 3CN) measurement result: d=7.37-7.44 (m, 6H), 7.63 (d, J (H, H)=6.0Hz, 1H), 7.69-7.78 (m, 5H), 8.0 (m, 5H), 8.49 (d, J (H, H)=8.0Hz, 5H), 8.57 (d, and J (H, H)=2.0Hz, 1H).ESI-MS(m/z)：749.1([M-PF 6] +)，302.0([M-2PF 6] 2+)。
The synthetic of ruthenium (II) (compound 2) closed in (2) two (2,2 '-bipyridine) (4-(3-nitro-4-amino-benzene oxygen)-2,2 '-bipyridine)
Under nitrogen protection with 38.5 milligrams of 3-nitros-4-amino-phenol (0.25mmol), 10 milligrams of sodium hydride (purity: 60%; 0.25mmol) stirring at normal temperature after 45 minutes in the dry acetonitrile of 30mL; add 89.4 milligrams of compounds 1 (0.1mmol), evaporated under reduced pressure solvent after stirring at normal temperature is spent the night.Thick product separated with silica gel column chromatography, with 100: 7: 0.5 CH 3CN-H 2O-KNO 3Solution is eluent.Decompression steams a spot of CH of product behind the solvent 3CN-H 2The O dissolving adds saturated phosphofluoric acid aqueous ammonium title complex is separated out.Filter collecting precipitation, and, get 91 milligrams of target compounds, productive rate: 90% after the vacuum-drying with the less water washing. 1HNMR (CD 3CN) measurement result: d=6.66 (s, 2H, NH2), 6.9 (m, 1H), 7.10 (d, J (H, H)=9.2Hz, 1H), 7.27 (m, 1H), 7.34-7.46 (m, 6H), 7.73 (m, 4H), 7.81 (d, J (H, H)=5.2Hz, 1H), 7.89 (d, J (H, H)=2.8Hz, 1H), 7.97-8.08 (m, 6H), 8.40 (d, J (H, H)=8.0Hz, 1H), 8.47-8.52 (m, 4H) .ESI-MS (m/z): 867.2 ([M-PF 6] +), 361.1 ([M-2PF 6] 2+).
(3) title complex [Ru (bpy) 2(dabpy)] (PF 6) 2Synthetic
The 10%Pd/C catalyzer that adds 50 milligrams in the 50mL ethanolic soln that contains 101.2 milligrams of compounds 2 stirred back adding 15 microlitre hydrazine hydrates and reflux 4 hours.Remove by filter catalyzer, the filtrate decompression evaporate to dryness.Products obtained therefrom is dissolved in a spot of CH 3CN-H 2Adding saturated phosphofluoric acid aqueous ammonium behind the O separates out title complex.Filter collecting precipitation, and, get 93.3 milligrams of target compounds, productive rate: 95% after the vacuum-drying with the less water washing. 1HNMR (CD 3CN) measurement result: d=6.33 (m, 1H), 6.42 (d, and J (H, H)=2.4Hz, 1H), 6.69 (d, and J (H, H)=8.4Hz, 1H), 6.81 (m, 1H), 7.34-7.45 (m, 6H), 7.69-7.76 (m, 4H), 7.81 (d, and J (H, H)=5.6Hz, 1H), 7.99-8.08 (m, 6H), 8.35 (d, J (H, H)=8.0Hz, 1H), 8.47 (m, 4H) .ESI-MS (m/z): 837.1 ([M-PF 6] +), 346.1 ([M-2PF 6] 2+).Results of elemental analyses: press C 36H 30F 12N 8OP 2Ru calculated value (%): C44.05, H3.08, N11.41; Measured value (%): C43.81, H3.42, N, 11.27.
(4) title complex [Ru (bpy) 2(T-bpy) 2+] (PF 6) 2Synthetic
Externally ice-water-bath cooling is down with 23 milligrams of [Ru (bpy) 2(dabpy)] (PF 6) 2(0.024mmol) be dissolved in 20 milliliters the 2mol/L hydrochloric acid.Stir the 0.5 ml water solution that adds 3.2 milligrams of Sodium Nitrites down, ice bath stirred 2 hours down.Reaction solution transfers to neutrality with the sodium hydroxide of 4mol/L, separates with silica gel column chromatography behind the evaporate to dryness, with 100: 10: 1 CH 3CN-H 2O-KNO 3Solution is eluent.Decompression is used a spot of CH of product after steaming solvent 3CN-H 2The O dissolving adds saturated phosphofluoric acid aqueous ammonium title complex is separated out.Filter collecting precipitation, and, get 11.2 milligrams of target compounds, productive rate: 47% after the vacuum-drying with the less water washing. 1HNMR (CD 3CN) measurement result: d=6.92 (m, 1H), 7.27 (d, and J (H, H)=8.4Hz, 1H), and 7.27-7.48 (m, 5H), 7.51 (d, J (H, H)=6.4Hz, 1H), 7.72 (m, 5H), 7.85 (d, and J (H, H)=5.2Hz, 1H), 7.95-8.09 (m, 7H), 8.35 (d, J (H, H)=8.0Hz, 1H), 8.47-8.51 (m, 4H) .ESI-MS (m/z): 848.1 ([M-PF 6] +), 351.4 ([M-2PF 6] 2+).Results of elemental analyses: press C 36H 27F 12N 9OP 2Ru calculated value (%): C43.56, H2.74, N12.70; Measured value (%): C43.06, H3.02, N, 12.23.
Embodiment 2: probe Ru (bpy) 2(dabpy) 2+And with NO reaction after product Ru (bpy) 2(T-bpy) 2+Property testing
(1) spectral quality
With the pH value 7.4 0.1mol/L borate buffer solution has been measured title complex as solvent uv-vis spectra, fluorescence spectrum, molar extinction coefficient (e) and fluorescent quantum yield (f).It is Perkin ElmerLambda 35 type spectrophotometers that uv-vis spectra is measured with instrument, the fluorometric assay instrument is a Perkin Elmer LS 50B spectrophotofluorometer, fluorescence quantum yield uses three (2,2 '-bipyridine) closes ruthenium (II) title complex and record (document 16:K.Nakamaru with literature method as standard substance, Bull.Chem.Soc.Jpn.1982,55,2697).Measurement result sees Table 1.
Table 1.Ru (bpy) 2(dabpy) 2+And Ru (bpy) 2(T-bpy) 2+Absorption in borate buffer solution and photoluminescent property
Compound maximum absorption wavelength e 455nmMaximum emission wavelength f
(nm) (cm -1mol -1L) (nm) (％)
Ru(bpy) 2(dabpy) 2+ 455 13000 610 0.13
Ru(bpy) 2(T-bpy) 2+ 455 9540 616 2.2
By table 1 as seen, probe Ru (bpy) 2(dabpy) 2+With its fluorescence quantum yield before and after the NO reaction very big variation has taken place, Ru (bpy) 2(dabpy) 2+Fluorescence quantum yield have only 0.13%, and Ru (bpy) 2(T-bpy) 2+Fluorescence quantum yield reached 2.2%, show probe Ru (bpy) 2(dabpy) 2+16.9 times have been increased with its quantum yield of NO reaction back.The fluorescence spectrum of two kinds of compounds as shown in Figure 2.
(2) the pH value of solution value is to Ru (bpy) 2(dabpy) 2+And Ru (bpy) 2(T-bpy) 2+The influence of photoluminescent property
With title complex Ru (bpy) 2(dabpy) 2+And Ru (bpy) 2(T-bpy) 2+Its fluorescence intensity under different pH values is measured in phosphate buffer solution dissolving back with the 0.1mol/L of different pH values, the results are shown in Figure 3.As seen from the figure, the pH value greater than 5 scope in, Ru (bpy) 2(dabpy) 2+And Ru (bpy) 2(T-bpy) 2+Fluorescence intensity be subjected to the influence of pH value little, show that this probe all can use in slightly acidic, neutrality and weakly alkaline environment.
(3) probe Ru (bpy) 2(dabpy) 2+Selectivity to NO mensuration
Investigated active specy H respectively 2O 2, OH, OCl -, singlet oxygen ( 1O 2), NO 2 -, NO 3 -, ONOO -, O 2 -And NO and probe Ru (bpy) 2(dabpy) 2+Response situation, same concentrations and reaction conditions (respond and all in the borate buffer solution of the pH of 0.1mol/L value 7.4, carry out under the room temperature, the reaction times is 1 hour, reaction density is: Ru (bpy) 2(dabpy) 2+: 10mmol/L; H 2O 2: 100mmol/L H 2O 2OH:100mmol/L H 2O 2+ 100mmol/L (NH 4) 2Fe (SO 4) 2OCl -: 100mmol/L NaOCl; 1O 2: 100mmol/L H 2O 2+ 100mmol/L NaOCl; NO 2 -: 100mmol/L NaNO 2NO 3 -: 100mmol/L NaNO 3ONOO -: 100mmol/L NaONOO; O 2 -: 100mmol/L KO 2NO:40mmol/L) following 9 kinds of active species and Ru (bpy) 2(dabpy) 2+The fluorescent strength determining result of reaction product as shown in Figure 4.As seen from the figure, probe Ru (bpy) 2(dabpy) 2+With H 2O 2, OH, OCl -, 1O 2, NO 2 -, NO 3 -, ONOO -, O 2 -Considerable change does not take place in reacted fluorescence intensity, shows Ru (bpy) 2(dabpy) 2+Do not react with these active species.As Ru (bpy) 2(dabpy) 2+After the NO reaction, owing to formed the Ru (bpy) of hyperfluorescence 2(T-bpy) 2+, cause the fluorescence intensity of probe significantly to strengthen.The above results shows probe Ru (bpy) 2(dabpy) 2+NO mensuration had good selectivity.
Embodiment 3: use Ru (bpy) 2(dabpy) 2+Measure the concentration of NO in the aqueous solution
In being 7.4 0.1mol/L borate buffer solution, the pH value adds Ru (bpy) respectively 2(dabpy) 2+(10mmol/L) and the NO of different concns, stirring reaction was measured fluorescence intensity after 0.5 hour.Measuring with instrument is Perkin Elmer LS 50B spectrophotofluorometer.
Shown in Fig. 5 (A), Fig. 5 (B), be accompanied by the increase of NO concentration, the fluorescence intensity of probe also increases gradually, shows Ru (bpy) 2(dabpy) 2+The concentration that can be used for the NO that generates in the quantitative assay solution.Fig. 6 has provided use Ru (bpy) 2(dabpy) 2+Detect the working curve of NO.As shown in the figure, the concentration of NO and fluorescence intensity have good linear relationship, and calculating minimum detectabilities with 3 times of the background signal standard deviation is 0.27mmol/L, show and use Ru (bpy) 2(dabpy) 2+Detection by quantitative NO has higher sensitivity.
Embodiment 4: probe Ru (bpy) 2(dabpy) 2+The fluorescence imaging that is used for NO in the viable cell is measured
(1) Ru (bpy) 2(dabpy) 2+The fluorescence imaging of exogenous NO is measured in the zooblast of mark
With [the Ru (bpy) that newly prepares 2(dabpy)] (PF 6) 2DMSO solution be diluted to the stock solution that concentration is 1.0mmol/L (DMSO concentration is 0.1%) with the DMEM substratum.With this water culture mouse macrophage, at 5%CO 2Cultivate after 5 hours down for 37 ℃ in the incubator, discard nutrient solution and with normal saline solution solution thorough washing cell to remove the probe molecule that does not enter cell, in culturing bottle, add 1-hydroxyl-2-oxygen-3-(3-the aminopropyl)-3-methyl isophthalic acid-triazene that contains 1.0mmol/L and (be called for short NOC13, its hydrolyzable in the aqueous solution produces NO) grade ooze salts solution, at 5%CO 2Cultivate under 37 ℃ in the incubator and carry out fluorescence imaging mensuration after 0.5 hour.
Fig. 7 has provided the cell fluorescence image that does not add NOC13 and add NOC13, as seen from the figure, when not adding NOC13, Ru (bpy) 2(dabpy) 2+The scavenger cell of mark does not almost have fluorescence, can obviously observe the fluorescence that cell sends after adding NOC13.Fluorescence intensity distribution situation by pair cell is analyzed, the fluorescence intensity of finding tenuigenin and nucleus place is basic identical, show that probe molecule can permeate through cell membranes enter tenuigenin and enter into nucleus, and then everywhere NO carries out fluorescent microscopic imaging and measures in the pair cell.
(2) Ru (bpy) 2(dabpy) 2+The fluorescence imaging of endogenous NO is measured in the vegetable cell of mark
Contain 500mmol/L Ru (bpy) with joining after the cape jasmine cell filtration collection in cultivating 2(dabpy) 2+Grade ooze in the salts solution that to form density be every milliliter 3.5 * 10 5The suspension of cell, at room temperature shaking culture.Got a certain amount of enchylema every 0.5 hour and after centrifugal 5 minutes, ooze the salts solution thorough washing with grade and remove the title complex that does not enter cell, the cell after the washing is placed carry out fluorescence imaging mensuration on the slide glass at 4 ℃ of following 400rpm.
Fig. 8 has provided the fluorescence imaging measurement result of cape jasmine cell under the different incubation times, and as seen from the figure, along with the increase of incubation time, the fluorescence that cell sends also obviously strengthens, and shows in culturing process sustainable generation NO in the cape jasmine cell.Fluorescence intensity by pair cell is analyzed, and finds that the fluorescence intensity of nuclear area will illustrate that the generation of NO in the vegetable cell occurs in nuclear area apparently higher than the tenuigenin zone.
Use same condition with Ru (bpy) 2(T-bpy) 2+Cultivate with the cape jasmine cell and to carry out fluorescence imaging after 5 hours and measure, the result as shown in Figure 9.At this moment cell does not almost have fluorescence radiation, and Ru (bpy) is described 2(T-bpy) 2+Can not pass cytolemma and enter in the cell, illustrate that also probe enters the product Ru (bpy) that generates with the NO reaction behind the cell 2(T-bpy) 2+Can not pass cytolemma and be diffused in the nutrient solution, can not in the cell washing process, be washed out.

Claims (3)

1. nitric oxide fluorescent probe based on ruthenium (II) title complex is characterized in that: be the title complex that forms with divalent ruthenium and bipyridine and derivative thereof, wherein said complex structure formula is:
Figure A2009103082590002C1
2. the application of the described nitric oxide fluorescent probe of claim 1, it is characterized in that: in all kinds of biologies, microorganism and abiotic system, utilize described ruthenium (II) coordination compound fluorescent probe under containing oxygen condition with system in the NO specific reaction, make the fluorescence radiation of probe significantly strengthen, determine generation, growing amount and the distribution situation of NO then by fluorometry; Described fluorometry comprises conventional fluorescent spectrophotometer assay method or fluorescent microscope imaging assay method.
3. application according to claim 2 is characterized in that: described determination of nitric oxide is used for containing the mensuration test kit and the related reagent of its component with ruthenium (II) coordination compound fluorescent probe.
CN200910308259A 2009-10-14 2009-10-14 Nitric oxide fluorescent probe based on ruthenium (II) complex and application thereof Pending CN101671555A (en)

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CN103421486A (en) * 2012-05-22 2013-12-04 中国科学院理化技术研究所 Fluorescence probe, synthetic method therefor and applications
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CN103073538A (en) * 2012-12-31 2013-05-01 中国食品药品检定研究院 Application of 1,4-dihydropyridine derivative as NO fluorescent probe
CN103073538B (en) * 2012-12-31 2015-05-20 中国食品药品检定研究院 Application of 1,4-dihydropyridine derivative as NO fluorescent probe
CN103923641A (en) * 2014-05-06 2014-07-16 辽宁大学 Fluorescent probe for detecting nitric oxide in mitochondria and application of fluorescent probe
CN103923641B (en) * 2014-05-06 2015-08-12 辽宁大学 Nitric oxide production fluorescent probe and application thereof in a kind of detection line plastochondria
CN104650334A (en) * 2015-02-16 2015-05-27 上海交通大学 Preparation method of degradable necklace-like nitric oxide fluorescent macromolecular probe
CN104650334B (en) * 2015-02-16 2016-08-24 上海交通大学 The preparation method of degradable item chain nitric oxide fluorescence polymer probe
CN106645046A (en) * 2016-09-18 2017-05-10 北京工业大学 Method for in-situ determination of intracellular nitric oxide (NO) of microbes in denitrified activated sludge

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