CN110128414A - A kind of preparation and application of the anoxic fluorescence probe based on hemicyanine dye - Google Patents
A kind of preparation and application of the anoxic fluorescence probe based on hemicyanine dye Download PDFInfo
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The preparation and application of the present invention relates to a kind of anoxic fluorescence probe based on hemicyanine dye, the structural formula of the fluorescence probe are as follows:The present invention provides the preparation methods for taking Hemicyanine fluorescent dye, sodium nitrite, phenol etc. as the Material synthesis fluorescence probe；The fluorescence probe is a kind of anoxic fluorescence probe near infrared emission, highly selective；Due to Na2S2O4It has been found to be used to simulate the anoxic reducing environment in biological sample, so we select Na2S2O4To replace external anaerobic environment.Firstly, the fluorescence probe is to Na2S2O4Show higher signal-to-noise ratio, probe and Na2S2O4Fluorescence significantly increases after reaction；Secondly, the fluorescence probe is to Na2S2O4Good selectivity is shown, not by the interference of other ions and reducing substances；In addition, the fluorescence probe has been successfully applied to cell imaging research, it can detecte intracellular anoxic conditions.
Description
Technical field
The invention belongs to fluorescent probe technique fields, and in particular to a kind of anoxic fluorescence probe based on hemicyanine dye
Preparation and application.
Background technique
Anoxic is malignant tumour there is a phenomenon where generally existing in, development process, generates and mainly gives birth to tumour without limitation
Long, oxygen consumption increase and tumor blood vessels depauperation etc. are related, and the pathologic process that clinical a variety of diseases are shared
(A.L.Harris,Nat.Rev.Cancer.,2002,2,38-47).Tumour cell under anaerobic environment easily shifts, and
Can increase the fastness to radiotherapy, chemotherapy, thus reduce therapeutic effect (L.J.Walker, R.B.Craig, A.L.Harris,
I.D.Hickson,Nucl Acids Res,1994,22,4884-4889；R.E.Durand,Int J Radiat Oncol
Biol Phys,1991,20,253-258；R.E.Durand,In Vivo,1994,8,691-702).Therefore, design is effective
Method goes the accurate occurrence and development rule for monitoring anoxic, and it is very important.
In recent years, fluorescence probe is because of the imaging of its high sensitivity in biopsy samples, real-time detection and high-spatial and temporal resolution
Outstanding advantages of and be concerned (H.Kobayashi, M.Ogawa, R.Alford, P.L.Choyke, Y.Urano,
Chem.Rev,2010,110,2620-2640.).Up to the present, the fluorescence probe for having developed some detection anoxics, is used
In real-time monitoring cell or intravital anoxic conditions (L.J.O ' Connor, I.N.Mistry, S.L.Collins,
L.K.Folkes,G.Brown,S.J.Conway,E.M.Hammond,ACS Cent.Sci.,2017,3,20-30；J.Zhang,
H.W.Liu,X.X.Hu,J.Li,L.H.Liang,X.B.Zhang,et al,Anal Chem.,2015,87,11832-11839；
K.K.Kiyose,K.Hanaoka,D.Oushiki,T.Nakamura,M.Kajimura,M.Suematsu,et al,J Am
Chem Soc.,2010,132,15846-15848；W.Piao,S.Tsuda,Y.Tanaka,S.Maeda,F.Liu,
S.Takahashi,et al,Angew Chem Int Ed,2013,52,13028-13032).But these anoxic fluorescence are visited
There is shortcomings for needle: (1) the transmitted wave length of fluorescence probe, is easy the interference by itself background fluorescence, is unfavorable for depth
Layer tissue image checking.(2) recognition group of probe is quinonyl or nitro, due to the presence of photo induced electron transfer mechanism (PET),
It is easy to be influenced by conditions such as pH or polarity.Therefore a kind of long wavelength emission is designed, there is the fluorescence of hypersensitivity to anoxic
Probe is vital.
Hemicyanine dye has been widely used in fluorescent probe technique field, it has big molar absorption coefficient, height
The advantages such as fluorescence quantum yield, it is most important that there is near infrared emission performance.Near infrared emission can penetrate deeper tissue,
It is not easily susceptible to the interference of organism itself fluorescence, it is more advantageous to bio-imaging.It is reported that having been succeeded using half flower cyanines fluorescence probe
Many objects are had detected, such as: ALP, CO, H2O2Deng (S.J.Li, C.Y.Li, Y.F.Li, J.J Fei, P, Wu, B, Yang,
J.Ou-Yang,S.X.Nie,Anal.Chem.2017,89,6854-6860；S.J.Li,D.Y.Zhou,Y.F.Li,B.Yang,
J.Ou-Yang,J.Jie,J.Liu；C.Y.Li,Talanta 2018,188,691-700；L.Yuan,W.Lin,S.Zhao,
W.Gao,B.Chen,L.He,S Zhu,J.Am.Chem.Soc.,2012,134,13510-13523).But currently not yet
Anoxic is detected based on the fluorescence probe of hemicyanine dye.Therefore, the near-infrared fluorescent that design synthesizes a kind of hemicyanine dye is visited
Needle is necessary as the effective tool of degree of oxygen deficiency in detection biological sample.
Summary of the invention
According to proposed requirement, the present inventor has made intensive studies this, after having paid a large amount of creative works,
Provide a kind of anoxic fluorescence probe based on half flower cyanines.
The technical scheme is that a kind of anoxic fluorescence probe based on hemicyanine dye, structural formula are as follows:
A kind of preparation method of the anoxic fluorescence probe based on hemicyanine dye.Steps are as follows:
In 100mL round-bottomed flask, 10mL is added and contains 1 equivalent Cy-NH2Volume ratio be 1:1 CH3CN/CH2Cl2It is mixed
Solution is closed, under -5-5 DEG C and nitrogen protection, after the concentrated hydrochloric acid of 3.5 equivalents is added dropwise, is stirred evenly.Then, 1 equivalent will be contained
NaNO2Distilled water solution be slowly dropped in above-mentioned reaction solution, continue stir 20-30min.Then, it is added and contains 2 equivalent ammonia
The aqueous solution of base sulfonic acid continues to stir 5-10min.Then, the acetonitrile solution containing 6 equivalent of phenol is added in above-mentioned reaction solution
And pH is adjusted as 6.0-6.5, under similarity condition, continue to stir 1.5-2h.After the reaction was completed, it is diluted with water and uses CH2Cl2Extraction,
Collected organic layer is washed with brine, anhydrous Na2SO4It is dried, filtered and concentrated.The CH that crude product is 20:1 with volume ratio2Cl2/
CH3OH eluant, eluent carries out column chromatography, obtains greenish solid product Cy-AP (yield 48%), as the fluorescence probe.
The invention has the advantages that a kind of good spectral response of the anoxic fluorescence probe based on hemicyanine dye
Energy.Na2S2O4Since its strong reducing property is already used in simulation tumour cell because of anoxic existing reducing environment, institute
Na is selected with us2S2O4Detectable substance as detection degree of oxygen deficiency.Firstly, studying the fluorescent spectroscopic properties of the probe.It is added
Na2S2O4Before, fluorescence probe does not have the fluorescence emission peak of near-infrared；Na is added2S2O4Later, occur at 725nm close red
External emission peak.And with Na2S2O4The near-infrared fluorescent intensity of the increase of concentration, probe constantly enhances.Then, probe is studied
Ultra-violet absorption spectrum.In no addition Na2S2O4When, probe has a higher absorption peak at 450nm, has at 650nm
One lesser absorption peak；Na is added2S2O4Afterwards, the absorption peak at 450nm is gradually reduced, and occurs new strong suction near 680nm
Receive peak.Then, the selectivity of probe is studied.Investigate probe and various ion (Na+,K+,Ca2+,Mg2+,Fe2+,ClO-,OH-,Br-,
I-,F-) and some amino acid (Lysine, Tryptophan, Leucine, Glycine, Alanine) and detectable substance
(Na2S2O4) fluorescence response situation.As a result, it has been found that only Na2S2O4It can cause the change of fluorescence spectrum, other detectable substances are to spy
The fluorescence spectrum of needle does not change significantly.Finally, fluorescence probe response is rapider, the response time is within 15 minutes.
A kind of application of the anoxic fluorescence probe based on hemicyanine dye.Cell is added glimmering after normal oxygen environment culture
Light probe, almost without the generation for observing fluorescence.After anaerobic environment culture probe is added, it can be seen that fluorescence in cell
It is remarkably reinforced.Moreover, with the intensification of degree of oxygen deficiency, fluorescence intensity is gradually increased.These results illustrate fluorescence probe Cy-AP energy
The variation of intracellular degree of oxygen deficiency is monitored, this provides a kind of reliable for the anoxic conditions during related pathologies in monitor's body
Means.
Detailed description of the invention
Fig. 1 is the synthetic route of fluorescence probe.
Fig. 2 is the Na of fluorescence probe and various concentration2S2O4Fluorescence spectra after effect.
Abscissa is wavelength, and ordinate is fluorescence intensity.The concentration of fluorescence probe is 10 μM, Na2S2O4Concentration difference
Are as follows: 0,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0mM.Launch wavelength is that the corresponding excitation wavelength of 725nm is
680nm。
Fig. 3 is fluorescence probe and Na2S2O4Act on the ultraviolet-visible absorption spectroscopy figure of front and back.
Abscissa is wavelength, and ordinate is absorbance.The concentration of fluorescence probe is 10 μM, Na2S2O4Concentration is 5.0mM.
Fig. 4 is the selective figure of fluorescence probe.
The concentration of fluorescence probe is 10 μM, and 1-16 analyte is not to be: 1, Na+；2,K+；3,Ca2+；4,Mg2+；5,Fe2+；
6,ClO-；7,OH-；8,Br-；9,I-；10,F-；11,Lysine；12,Tryptophan；13,Leucine；14,Glycine；15,
Alanine；16,Na2S2O4。
Fig. 5 is fluorescence probe and Na2S2O4The graph of relation that fluorescence intensity changes over time after effect.
Fig. 6 is cytotoxicity experiment figure.Abscissa is the concentration of fluorescence probe, and ordinate is the survival rate of cell.
Fig. 7 is cell hypoxia fluorescence imaging figure.Four kinds of cells (HepG2 cells, HCT116 cells, HeLa cells,
MCF-7 cells) 1h is cultivated in different anaerobic environments, then probe dyes 0.5h.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings, but not limited to this.
Embodiment 1:
The synthesis of fluorescence probe
Synthetic route such as Fig. 1.In 100mL round-bottomed flask, 10mL is added and contains Cy-NH2(0.17g, 0.3mmol's)
CH3CN/CH2Cl2(1:1) mixed solution is added concentrated hydrochloric acid (0.05mL, 1mmol), stirs evenly under 0 DEG C and nitrogen protection.
Then, NaNO will be contained2The distilled water solution of (0.02g, 0.3mmol) is slowly dropped in above-mentioned reaction solution, continues to stir
30min.Then, the aqueous solution of sulfamic acid (0.06g, 0.6mmol) is added, continues to stir 10min.Then, phenol will be contained
The CH of (0.16mL, 1.8mmol)3CN solution is added in reaction solution, and adjusting pH is 6.0, under similarity condition, continues to stir
1.5h.After reaction, it is diluted with water, uses CH2Cl2Extraction.Collected organic layer is washed with brine, anhydrous Na2SO4It is dry, mistake
It filters and is concentrated.The CH that obtained crude product is 20:1 with volume ratio3OH/CH2Cl2Eluent carries out column chromatography, and it is solid to obtain bottle green
Body Cy-AP (0.08g), yield 48%, as fluorescence probe.1H NMR(400MHz,DMSO):δ10.62(s,1H),8.71(d,
J=15.2Hz, 1H), 8.41 (d, J=8.4Hz, 1H), 8.22-8.14 (m, 2H), 7.98 (d, J=8.8Hz, 1H), 7.86-
7.65 (m, 6H), 7.41 (s, 1H), 6.98 (d, J=8.8Hz, 2H) .6.71 (d, J=15.6Hz, 1H), 4.60 (d, J=
7.2Hz, 2H), 1.84 (s, 2H), 1.43 (t, J=6.4Hz, 4H), 1.191 (s, 6H), 0.80 (t, J=4.8Hz, 3H)13C
NMR(100MHz,DMSO):δ179.7,158.9,153.8,153.3,145.8,145.0,139.0,137.3,133.1,
131.8,131.4,130.1,128.8,128.7,127.5,127.0,125.9,123.9,123.3,119.7,116.7,
115.2,113.0,109.4,106.4,53.0,40.8,29.3,27.3,24.1,22.6,20.3,13.7.MS(TOF):
552.5.
Embodiment 2:
Fluorescence probe and Na2S2O4Solution is prepared
The preparation of probe solution: it weighs a certain amount of probe solid dissolution in methyl alcohol, is made into 1 × 10-4The probe solution of M.
Na2S2O4The preparation (matching while using) of solution: a certain amount of Na is weighed2S2O4Solid is dissolved in distilled water, is made into 1 × 10-1The inspection of M
Survey solution.By 1.0 × 10-1The Na of M2S2O4Solution gradually dilutes, and obtains 1.0 × 10-1-1.0×10-3The Na of M2S2O4Solution.It will
The stock solution of 1.0mL probe and the Na of 1.0mL2S2O4Solution is added in the volumetric flask of 10mL, after buffer solution constant volume,
Obtaining concentration is 1.0 × 10-5The fluorescence probe of M and 1.0 × 10-2-1.0×10-4The Na of M2S2O4Mix solution to be measured.
Embodiment 3:
Fluorescence probe and Na2S2O4The measurement of the fluorescence spectrum of effect
Fig. 2 is fluorescence probe and Na2S2O4The fluorescence spectrum of effect, the concentration of fluorescence probe are 10 μM, Na2S2O4Concentration
It is followed successively by 0,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0mM.Testing excitation wavelength used is 680nm, transmitted wave
Long range is 705~800nm.Slit width is 10.0nm/10.0nm, and fluoremetry instrument used is Hitachi F4600 fluorescence
Spectrophotometer.Figure it is seen that Na is added2S2O4Before, due to the quenching effect of azo bond, fluorescence probe does not have fluorescence
Emission peak；Na is added2S2O4Later, near infrared region, there is near-infrared fluorescent emission peak in (725nm).This is because probe point
Son is by Na2S2O4Reduction, leads to the fracture of azo bond, half flower cyanines fluorogen is released, to generate near-infrared fluorescent.And with
Na2S2O4The near-infrared fluorescent intensity of the increase of concentration, probe molecule constantly enhances.
Embodiment 4:
Fluorescence probe and Na2S2O4The measurement of the ultraviolet-visible absorption spectroscopy of effect
Fig. 3 is fluorescence probe and Na2S2O4Ultraviolet-visible absorption spectroscopy figure after effect, the concentration of fluorescence probe are 10 μM,
Na2S2O4Concentration be 5.0mM.The instrument of ultraviolet-visible absorption spectroscopy measurement is Agilent Cary60 UV, visible light light splitting light
Degree meter.From figure 3, it can be seen that in no addition Na2S2O4When, probe has higher absorption peak at 450nm, at 650nm
There is lesser absorption peak；Na is added2S2O4Afterwards, the absorption peak at 450nm fades away, and occurs new strong absorption at 680nm
Peak.
Embodiment 5:
Fluorescence probe is to Na2S2O4The selectivity of measurement
Fig. 4 is fluorescence probe to Na2S2O4The selective figure of measurement.It investigates in the fluorescence probe solution that concentration is 10 μM
Na is added2S2O4(5.0mM) and various ion (Na+,K+,Ca2+,Mg2+,Fe2+,ClO-,OH-,Br-,I-,F-) and some ammonia
The fluorescence response situation of base acid (Lysine, Tryptophan, Leucine, Glycine, Alanine).It can from Fig. 4
Out, only Na2S2O4It can cause the change of fluorescence spectrum, other detectable substances do not influence the fluorescence spectrum of probe significantly.This
The result shows that, fluorescence probe is to Na a bit2S2O4There is preferable selectivity.
Embodiment 6:
Fluorescence probe and Na2S2O4The measurement of the response time of effect
We have studied fluorescence probes to Na2S2O4Response time, result such as Fig. 5.It can be seen from the figure that the spy
For Na2S2O4Response time be 15min, this can satisfy in actual sample carry out real-time monitoring when to the response time
It is required that.After from Fig. 5, we can also be seen that fluorescence intensity reaches maximum value, as time increases, fluorescence intensity no longer occurs
Variation, this shows that this fluorescence probe photostability is preferable.
Embodiment 7:
Application of the fluorescence probe in living cells
Firstly, we have done cytotoxicity experiment, as shown in Figure 6.After 0~30 μM of Cy-AP is added, four kinds of cells
The survival rate of (HepG2 cells, HCT116 cells, HeLa cells, MCF-7 cells), therefore can 90% or more
With explanation, the fluorescence probe toxicity is smaller.Then, we study application of the fluorescence probe in living cells, select HepG2
Tetra- kinds of tumour cells of cells, HCT116 cells, HeLa cells, MCF-7 cells carry out confocal microscopic image, as a result
As shown in Figure 7.By normal oxygen culture (20%O2) cell, almost without the generation for observing fluorescence.Four kinds of cells are in advance not
With degree anoxic (10%O2, 5%O2, 1%O2) culture 1h, after then dyeing 0.5h with probe, fluorescence is remarkably reinforced.And with
The raising of degree of oxygen deficiency, fluorescence intensity be gradually increased.These results illustrate that fluorescence probe can monitor intracellular degree of oxygen deficiency
Variation, this provides a kind of reliable means for the anoxic conditions during related pathologies in monitor's body.
Claims (3)
1. a kind of anoxic fluorescence probe based on hemicyanine dye, i.e. Cy-AP, structure are as follows:
2. a kind of preparation method of anoxic fluorescence probe based on hemicyanine dye according to claim 1, feature exist
In reaction step is as follows:
In 100mL round-bottomed flask, 10mL is added and contains 1 equivalent Cy-NH2Volume ratio be 1:1 CH3CN/CH2Cl2It mixes molten
Liquid is added dropwise the concentrated hydrochloric acid of 3.5 equivalents, stirs evenly under -5~5 DEG C and nitrogen protection；Then, 1 equivalent NaNO will be contained2's
Distilled water solution is slowly dropped in above-mentioned reaction solution, continues 20~30min of stirring；Then, it is added and contains 2 equivalent sulfamic acids
Aqueous solution, continue 5~10min of stirring；Then, the acetonitrile solution containing 6 equivalent of phenol is added in above-mentioned reaction solution and is adjusted
Saving pH is 6.0~6.5, under similarity condition, continues 1.5~2h of stirring；After the reaction was completed, it is diluted with water and uses CH2Cl2Extraction is received
Collect organic layer, is washed with brine, anhydrous Na2SO4It is dried, filtered and concentrated, the CH that crude product is 20:1 with volume ratio2Cl2/
CH3OH eluant, eluent carries out column chromatography, obtains greenish solid product Cy-AP, the as fluorescence probe.
3. a kind of application of anoxic fluorescence probe based on hemicyanine dye according to claim 1, which is characterized in that institute
State the detection that fluorescence probe is applied to the variation of intracellular degree of oxygen deficiency.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111518546A (en) * | 2020-05-19 | 2020-08-11 | 遵义医科大学 | Hypoxic microenvironment response fluorescent probe and preparation method and application thereof |
| CN112047979A (en) * | 2020-09-10 | 2020-12-08 | 山东师范大学 | Fluorescent probe Mito-HNO, preparation method thereof and application thereof in detection of HNO in mitochondria |
| CN113354583A (en) * | 2021-06-15 | 2021-09-07 | 上海大学 | Fluorescent probe for detecting hypoxic level, preparation method and application thereof |
| CN114478493A (en) * | 2022-01-27 | 2022-05-13 | 中国科学院兰州化学物理研究所 | Traceable 5-aminosalicylic acid derivative and preparation and application thereof |
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| CN114478493B (en) * | 2022-01-27 | 2024-04-02 | 中国科学院兰州化学物理研究所 | Traceable 5-aminosalicylic acid derivative and preparation and application thereof |
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