Near infrared fluorescent probe for detecting zinc ion in cell and its synthesis method and use
Technical Field The present invention relates to biological detection technology and clinical medical detection field, especially relates to a device for detecting of zinc ion in the cell near-infrared fluorescence probe; the invention also relates to a method for the synthesis of the fluorescent probe; in addition, the invention also relates to the use of the fluorescent probe. Background Art In organisms of the zinc ion is a transition metal of enrichment is 2nd. A large amount of zinc ion in the concentrated in the nervous tissue, such as in the brain tissue concentration of 0.1-0.5 nm. Zinc ion through the metal regulating protein directly involved in regulating gene expression, such as zinc as a of the zinc finger protein, can be changed by changing chromalloy structure or by interrupting DNA synthesis to influence gene expression. In vivo the majority of the zinc ion in close combination with the protein, in the presence of certain cells "free zinc bath". Zinc ion in the metal protein as a kind of structure factor. Zinc can inhibit the generation of superoxide radical and the poison effects of the superoxide radical elimination. Synaptic zinc ion can be released from the pocket and through electric potential calcium ion channel and enter the cell, this shows that the free zinc ion with nerve regulating function. At the same time, a large amount of evidence within and outside the organism have indicated that zinc in apoptosis is an important element to adjust. Many of the existing method, including atomic absorption spectrometry, electron paramagnetic resonance, such as Probe Microanalysis Electron, has been used for a cell in the detection of metal ions, but these expensive instrument for by the method, can not be metal ions to the biological in vivo real-time, in-situ dynamic detection, and the method to determine the pre-treatment of the sample is relatively complicated, thereby, subject to certain restrictions. At present, existing cell zinc ion can be used for the detection of the fluorescent probe has the following categories: TSQ (Frederickson, C.J. ; Kasarkis, E.J. ; Ringo, D. ; Frederickson, R.E.J. Neurosci.Methods, 1987, 20, 91-103.), Zinquin b ester (Tsuda, M.Neurosci. , 17, 6678, 1997) pellet sulfonyl amino ethyl link amine (Koike, t. ; Watanabe, t. ; Aoki, S. ; Kimura, E. ; Shiro, m. , J.Am.Chem.Sic. , 118, 12696-12703, 1996) zinpyr (Burfrtte, S. C. ; Frederickson, C.J. ; Bu, W. ; Lippard, S.J. , J.Am.Chem.Soc. , 2003,125, 1778-1787. And ZnAF (Hirano, t. ; Kikuchi, K. ; Urano, Y. ; Higuchi, t. ; Nagano, T.J.Am.Chem.Soc. , 2002, 124, 6555-6562.), and the like, but these probes shortcoming lies in its excitation wavelength is in the ultraviolet region (-350 nm), when damage to the excitation of the cells, and because its emission wavelength in the ultraviolet or visible region, biological body self-fluorescence interference is serious, are sensitive to changes in pH, the restriction to a certain degree. In addition of this type of probe molecule synthesis is generally more difficult to, the structure is more complicated, there will be certain difficulties in practical application. Content of the invention The purpose of this invention to improve the existing for the fluorescent probe for zinc ion detection of the deficiency of the performance and structure, the design and synthesis of good performance, is suitable for the zinc ion detection of the fluorescent dyes, cyanine-based probe molecule as a research goal. One of the purposes of this invention aims at overcoming the prior art fluorescent probe performance and structural deficiencies, provides a good performance, is suitable for the zinc ion detection of the fluorescent dyes, cyanine-based detection of zinc ion in the cell near-infrared fluorescence probe; the second objective is to provide process is simple, low cost, convenient operation of the near-infrared fluorescent probe synthesis method; the third purpose is to provide the use of the fluorescent probe. One of the purposes of this invention can be realized by the following technical measures: The cyanine dye fluorescent probe has the following general structure: Formula: R=-CH2 CH3, -CH2 CH2 CH3, -CH2 CH2 CH2 CH2 SO3-. The second objective of the present invention can be realized by the following technical measures: This synthetic method is carried out according to the following steps: A. In the cyanine dyes Cy. 7 = 1 the complex radical [...][...] 5-10 to the weight proportion of the raw material by adding N, N-dimethyl formamide is dissolved, then the inert gas protection and 60-70 the reaction under [...] 2-5 hours; B. A process of the room temperature the resulting reaction mixture is cooled to under the 10-20 [...] , to dryness under reduced pressure, ethyl ether after dissolved, add water extraction to layered, extracting liquid I by adding ethyl acetate extraction to layered, to extract II; C. The b process the extraction liquid II drying with anhydrous magnesium sulfate, after the 30-40 under [...] to dryness under reduced pressure, evaporating ethyl acetate, to obtain the product. The second objective of the present invention can also be realized by the following technical measures: A process wherein the cyanine dye Cy. 7 is a near-infrared fluorescent spectrum of the dye colored cyanine class ; a process wherein the complexing group is selected from a two (2-pyridylmethyl) amine, quinoline sulfonamide or 4, 7, 10-trimethyl -1, 4, 7, 10-tetra nitrogen heterocyclic doecyl; b process the water: diethyl ether = 1 the [...] 1-1.5 the weight; said ethyl acetate: I=1 the extract [...] 1-1.5 the weight; said extraction is conducted all at room temperature; b, process c refer to decompress evaporation to dryness in the pressure of the oil pump, the 35-45 [...] rotary evaporation. Representative compounds of this invention displayed in the synthesis scheme below. Under the protection of inert gas, the cyanine fluorescent dye (2-pyridylmethyl) amine in N, N-dimethyl formamide in the probe molecule is obtained by reacting, finally through the extraction separation to obtain a pure product of the probe. Fluorescent probe of the present invention when in use there is no special restriction, usually, the probe molecule can be dissolved in physiological saline, buffer solution or by ethanol, acetonitrile, dimethyl sulfoxide, in the water-soluble organic solvent, then adding containing cell organization in the appropriate buffer solution for testing. The third purpose of the present invention can be realized by the following technical measures: The detection of this invention, the zinc ion in the cell near-infrared fluorescent probe used for chemical analog biological system the detection of the zinc ion, biological living cells and of zinc ions in living tissue detection and analysis of fluorescence imaging detection, pathological tissue and in the clinical medicine detection of zinc ion. Intracellular zinc ion detection is the general method for the culture of the cells is placed in a buffer solution containing probe molecules in, a part of the cell using a laser confocal microscope imaging blank Image, then a part of the remaining cells containing zinc ion and zinc ion-selective ion carrier, after flushing fluid are used, laser confocal microscopic imaging. Then will contain cells are immersed in a complex containing N, N, N ', N' -tetra (2-pyridylmethyl) ethylenediamine (TPEN) in the solution, laser confocal microscopic imaging. The invention has the following positive effects: The fluorescence probe molecule belonging to the near-infrared fluorescent probe, can effectively avoid the interference of the AUTOFLUORESCENCE of cells, improved selectivity and sensitivity of the detection method, to reduce the damage of the living body, the living body. Probe molecule stimulating the 720-730 range, transmitting in 780-790 range, is not sensitive to the polarity of the solvent, a larger Stokes displacement, and the chemical-optical stability is good; the fluorescence probe molecule of the invention in the molecule design is based on the principle of induced electron transfer (PET), fluorescence probe molecule complex zinc ion former fluorescence quantum yield is lower than 0.1, after complexing zinc ion fluorescence quantum yield is higher than 0.4, zinc ion complexation front and rear quantum yield at least 20 several times of growth; fluorescence probe molecule to the zinc ion has very good selectivity, sodium, potassium, calcium, magnesium, iron, cobalt, nickel, copper, mercury and other metal ion does not influence the detection; fluorescence probe molecule is not sensitive to changes in pH, when the pH value in the 6 to 8 the range, the pH value change does not influence of fluorescence-emission; fluorescence probe molecule complexed with the zinc ion between the constant in the micromolar to the picomole concentration range, can detect nanomolar concentration cell zinc ion; good permeability of cell fluorescence probe molecule, the cell itself has no toxic side effect, suitable for cell the zinc ion concentration change detection. The present invention relates to probe molecule has an extremely important application value. In particular, the series of probe molecule and its excitation wavelength lies in the near infrared region, there are very large Stokes displacement, is not sensitive to the pH change, the response time is extremely short, an extremely high measuring sensitivity, the cell permeability and small side effect, such probe as the correct measuring in vivo zinc ion reagent is extremely useful. Furthermore, the series of probe molecule the structure is simple, the synthetic method is simple and high in efficiency, to the actual application. Description of drawings Figure 1 is the the fluorescence probe molecule of the invention has good selectivity zinc ion; abscissa for various ion, ordinate to join metal ion of the probe molecule of metal ions the ratio of the fluorescence intensity of the solution. Fig. 2 is fluorescence probe molecule of the present invention and the fluorescent strength of the relationship between the concentration of the zinc ion; abscissa is a wavelength (nm) for the fluorescence intensity of the vertical. Figure 3 is the fluorescence probe molecule of the invention with the pH change relationship of the intensity of the fluorescent; abscissa as pH, ordinate is relative fluorescence intensity. Figure 4 is the study of the fluorescence probe molecule of the mouse macrophage (RAW264.7) laser confocal microscopic imaging. Mode of execution Reference with photos 1-4 further note: Probe to the zinc ion-selective Using the above-mentioned synthetic compound evaluation of zinc ion selectivity. The 1 the compound M applied to each kind of metal ion pH of 7.4 hydroxyethyl [...] b sulfur sulfonic acid buffer solution (0.1 mm), probe excitation wavelength for 731 nm, emission wavelength is 780 nm, the test result is shown in the Figure 2 in. Figure 1 in, the fluorescence intensity of the longitudinal axis, is not added the fluorescence intensity of the metal ions is 1, a numerical representation of the fluorescence intensity of the metal ions. In Figure 1 can be seen in, compound to the zinc ion has very high sensitivity, of the zinc ion to produce very great auxoflorence, in addition to the zinc ion also has very good selectivity, sodium, potassium, calcium, magnesium, iron, cobalt, nickel, copper, mercury and other metal ions has no influence on the detection. The pH sensitivity of the probe to the Using the above-mentioned synthetic compound evaluation of the sensitivity of the probe to the pH from Figure 3 it can be seen in in pH6 to 8 within the range, the pH does not basically influence of fluorescence-emission. Therefore, the probe can be used for the detection of the zinc ion under physiological conditions. Cell culture RAW264.7 by the sugar DMEM culture medium of the cell culture, prior to imaging, cell pastes the wall in cover wave on-chip, then add 1 the probe buffer M, 37 the incubating [...] 30 minutes, then a part of the cell using a laser confocal microscope imaging blank Image, then a part of the remaining cells containing zinc ion and zinc ion-selective ion carrier, after flushing fluid are used, laser confocal microscopic imaging. Then will contain complex cell is immersed in the solution containing TPEN, laser confocal microscopic imaging. Laser confocal imaging Using laser confocal microscope imaging technology, research of the mouse normal macrophage (RAW264.7) the zinc ion concentration changes in the impact of the fluorescence intensity to the probe. Through Figure 4 (a): probe incubation of macrophages, can see that a weak fluorescence; through Figure 4 (b): when the applied zinc ion, can be observed in the cytoplasm is of very strong fluorescent; through Figure 4 (c): on the basis of the added to the zinc ion has extremely strong after the complexing TPEN, fluorescence quenching; Figure 4 (d) is a bright field Image, to indicate that the cells in the whole experiment process of still maintaining good cell viability. Embodiment 1: Synthesis of probe In a 100 ml flask in three, by adding 0.3 g (5mmol) is provided with a near infrared fluorescent spectrum propyl cyanine dye Cy. 7 and 2 gram two (2-pyridylmethyl) amine, addition of about 60 ml N, N-dimethyl formamide to be completely dissolved, then in the nitrogen protection and 66 the reaction under [...] 4 hours, cooled to room temperature for the 15 [...] , after the pressure-reducing pump pressure-reducing, the 35 under [...] rotary evaporation to dryness, dissolved into ether cooled and liquid, then adding ether to the 1.2 times reacts by weight at room temperature of the extraction to layered, I the extract, then adding extraction of the liquid I 0.8 times parts by weight of the ethyl acetate extraction to layered, to extract II, finally to the extraction liquid II adding magnesium sulfate anhydrous drying, after the pressure-reducing pump pressure-reducing, the 35 under [...] rotary evaporation, evaporating ethyl acetate, to obtain the product 0.15 grams. Yield 50%. 1 H NMR (DMSO-d6): δ 0.97 (t, 6H, J=6.4Hz) 1.61 (m, 2H, J=5.5Hz) 1.68 (s, 12H) 1.72-1.82 (m, 4H) 2.73 (t, 4H, J=5.6Hz) 4.02 (s, 4H) 4.20 (t, 4H, J=6.8Hz) 6.34 (d, 2H, J=14.0Hz) 7.31 (t, 2H, J=7.32Hz) 7.48 (m, 4H) 7.64 (t, 2H, J=7.08Hz) 7.80-8.17 (m, 6H) 8.21 (d, 2H, J=14.0Hz) 8.66 (d, 2H, J=5.1Hz) ESI-MS cal for+ = 702.5, found 702.5. Embodiment 2: Quinoline sulfonamide replacement for the second (2-pyridylmethyl) amine, with the embodiment 1. Embodiment 3: With 4, 7, 10-trimethyl -1, 4, 7, 10-tetra nitrogen heterocyclic doecyl replacement b (2-pyridylmethyl) amine, with the embodiment 1. This invention is near-infrared fluorescent probe for intracellular zinc ion test. The syncetic process is as followings: A.mix cyanine dyes Cy.7 and complexing group in the weight propotion of 1:5-10, resolve the mix in N,N-dimethyl formamide, and react at 60-70deg C for 2-5 hours with the protection of inactive gas; B. Cool the production A to 10-20deg C, dry blowing with depression, resolve in aether, add water for extraction, the extraction liquor I obtained, then add acetic ester for extraction, the extraction liquor II obtained; C. Add anhydrous addex-magnesium in liquor II for desiccation, dry blowing with depression at 30-40deg C, get rid of acetic ester for the final production. The near-infrared fluorescent probe has a good cellular osmosis and little side effect. In particular, it's seligible for cellular zinc ion test. 1, the detecting zinc ion in cell near-infrared fluorescent probe, which is characterized in that the near infrared fluorescent probe is fluorescent probe cyanine dye, the cyanine dye fluorescent probe has the following general structure: Formula: R=-CH2 CH2 CH3. 2, claim 1 of zinc ions in the cell detection of the near-infrared fluorescent probe synthesis method, which is characterized in that the synthetic method is carried out according to following steps: A. In order to have near-infrared fluorescence spectrum propyl cyanine dye Cy. 7 the two complexing groups [...] (2-pyridylmethyl) amine = 1 the [...] 5-10 to the weight proportion of the raw material by adding N, N-dimethyl formamide is dissolved, then the inert gas protection and 60-70 the reaction under [...] 2-5 hours; B. A process of the room temperature the resulting reaction mixture is cooled to under the 10-20 [...] , to dryness under reduced pressure, ethyl ether after dissolved, add water extraction to layered, extracting liquid I by adding ethyl acetate extraction to layered, to extract II; C. The b process the extraction liquid II drying with anhydrous magnesium sulfate, after the 30-40 under [...] to dryness under reduced pressure, evaporating ethyl acetate, to obtain the product. 3, synthetic method according to Claim 2, characterized in that said water process b: diethyl ether = 1 the [...] 1-1.5 parts by weight of the; said ethyl acetate: I=1 the extract [...] 1-1.5 the weight; said extraction is conducted all at room temperature.