CN108033907A - A kind of Heptamethine cyanines active fluoro probe and preparation method and application - Google Patents

A kind of Heptamethine cyanines active fluoro probe and preparation method and application Download PDF

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CN108033907A
CN108033907A CN201711122247.0A CN201711122247A CN108033907A CN 108033907 A CN108033907 A CN 108033907A CN 201711122247 A CN201711122247 A CN 201711122247A CN 108033907 A CN108033907 A CN 108033907A
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probe
fluorescence
preparation
cyanines
heptamethine
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黄平升
王伟伟
秦怡博
孔德领
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Institute of Biomedical Engineering of CAMS and PUMC
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Abstract

The present invention relates to a kind of Heptamethine cyanines active fluoro probe and preparation method and application.General structure is as follows：In general formula:X=Ⅱ‑Ⅸ；R1、R2=(CH2)mCH3、(CH2)nOH、(CH2CH2O)pCH3、CH2C6H5；R3、R4=H、SO3H、SO3Na、SO3K；a‑g=2‑8；n、m、p=1‑10.The present invention is the Heptamethine cyanines fluorescent dye based on near-infrared long wave, selection indoline improves fluorescence intensity for fragrant parent nucleus, cyclohexene rigidity bridging enhances stability among methine chain, at the same time nucleophilic displacement of fluorine is carried out in seven methine cyanines parent dye middle positions using the azepine derivatives with chemical reactivity site, substantially increase Stokes shift, the advantages that active chemical group is easy to polytype material fluorescent marker, the fluorescence probe is symmetrical structure, preparation and purifying technique are simplified, advantageously reduces cost.The present invention can be used as the fluorescence labeling probe of the biomolecule such as highly sensitive albumen, sugar, DNA and nano-carrier, carry out cell or the imaging of live body level fluorescence.

Description

A kind of Heptamethine cyanines active fluoro probe and preparation method and application
Technical field
The present invention relates to a kind of Heptamethine cyanines active fluoro probe and preparation method and application, is specifically this kind of support The fluorescent marker for being suitable for the biomolecule such as highly sensitive albumen, sugar, DNA and nano-carrier of gram this displacement more than 100nm it is glimmering Light probe, carries out cell or the imaging of live body level fluorescence.
Background technology
The fluorescent marker of biomolecule is the quickly and easily biomolecule analysis means occurred after isotope marks.With The development of life science, there is an urgent need to highly sensitive analyzing detecting method.Medically, the enzyme using fluorescent technique as core Promoting immunity analytic approach and fluorescence polarization immunoassay have become the standard method of medical diagnosis.On molecular biology, utilize Laser confocal microscope tracks the position and movement situation of cellular content.In addition, flow cytometer is using fluorescent technique as core The heart carries out the discrimination and classification of cell.As nanosecond science and technology are in the application of field of medicaments, targeted nano fluorescence probe is utilized to carry out Diagnosing tumor treatment is as the hot spot studied.The exploitation of fluorescent dye be develop fluorescence analysis it is most critical because Element.
At present using more rhodamine, fluoresceins, BODIPY fluorochromes, its maximum absorption wavelength is all ultraviolet Visual field, and some biological samples have very strong absorption in this region, when fluoroscopic examination, cause very strong fluorescence background, significantly Reduce the sensitivity and accuracy of detection.The Absorption and emission spectra area of polymethin cyanine dyestuff is located at 550-1200nm (near red Outer fluorescence λem>600nm), near infrared region.Relative to conventional fluorescent probe (λem<For 600nm), in near-infrared fluorescent light Area, the fluorescence background of glass, polyacrylamide gel matrix and biochemistry impurity is very weak, and signal-to-noise ratio is high, and background interference is significantly Reduce.And they have very high molar absorption coefficient and good fluorescent characteristic.And due to scattered light intensity and wavelength Biquadratic be inversely proportional, with the increase of wavelength, Raman scattering is reduced rapidly, make scattering interference be also greatly lowered, therefore fluorescence examine The sensitivity of survey is very high.
Seven methine cyanine dyes are directed to as the focus that very important near infrared fluorescent probe is biomedical applications, forefathers The problems such as photostability and poorly water-soluble of this kind of dyestuff, has carried out the optimization in many structures.Mainly it is summarized as follows：(1) both ends It is best as fragrant parent nucleus performance using indoline；(2) Gabor Patonay etc. introduce rigid cyclohexene on methine chain, Photostability caused by solving the problems, such as methine chain elongation declines, and strengthens fluorescence intensity；(3) AS Wangger etc. are in molecule Middle introducing sulfonic group, improves stability and stability；(4) Peng Xiaojun etc. substitutes seven methine dyes middle position azepine derivatives, makes Intramolecular electron transfer effect occurs in excitation state in it, produces big Stokes shift, solves traditional seven methine dyes self-quenchings Problem.
But application of the seven methine indoles fluorescence probe of near-infrared long wave in terms of fluorimetric assay for biological materials at present it is still pure Some problems.
First, seven methine cyanines fluorescence probes of fluorescent marker can be carried out by chemically reacting, usually carry carboxyl or amber Amber acid imide active ester, and avtive spot is located on indoline fragrance parent nucleus, generally dissymmetrical structure, greatly increase synthesis The difficulty of complexity and separating-purifying.
Secondly, it is single that seven methenyl fluorochrome avtive spot species are commercialized at present, it is impossible to meet the biologies such as albumen, DNA The mark of bioactive molecule, polymer or inorganic nano carrier needs.
Again, commercialized seven methines cyanines fluorescence probe is expensive, it is necessary to optimum synthesis route and purifying technique at present.
The content of the invention
It is an object of the invention to provide a kind of new Heptamethine cyanines active fluoro probe and preparation method and application, The defects of prior art can be overcome.The present invention is the Heptamethine cyanines fluorescent dye based on near-infrared long wave, selects indoline Fluorescence intensity is improved for fragrant parent nucleus, cyclohexene rigidity bridging enhances stability among methine chain, passes through indoline parent nucleus The water solubility of upper sulfonic group regulation and control probe.At the same time using the azepine derivatives with chemical reactivity site in seven methine cyanines parents Dyestuff middle position carries out nucleophilic displacement of fluorine, substantially increases Stokes shift, active chemical group is easy to polytype material fluorescence mark It is the advantages that note, for biomolecule or the fluorescent marker of nano-carrier, fluorescence spectrum performance and cellular level and live body is horizontal Upper fluorescence imaging is as research emphasis.The fluorescence probe is symmetrical structure, greatly reduces the complexity of synthesis, simplifies preparation And purifying technique, advantageously reduce cost.The present invention can be used as the biomolecule such as highly sensitive albumen, polysaccharide, DNA and organic-inorganic The fluorescence probe of nano-carrier mark, carries out cellular level and detection and imaging in live body level.
Heptamethine cyanines active fluoro probe structure general formula provided by the invention is as follows：
In general formula:
X=II-Ⅸ；
R1、R2=(CH2)mCH3、(CH2)nOH、(CH2CH2O)pCH3、CH2C6H5；
R3、R4=H, SO3H、SO3Na、SO3K；
A-g=2-8；
N, m, p=1-10.
Wherein, a-g, n, m, p are mutually independent integer.
The preparation method of new Heptamethine cyanines active fluoro probe provided by the invention is by following step：
1) first, starting material indoline and alkylating reagent (iodoethane or benzyl bromide a-bromotoluene) are under argon gas protection, with toluene Flow back for solvent, quaterisation occurs, generates alkyl-substituted indoles quaternary ammonium salt intermediate, wherein indoline and alkyl The molar ratio for changing reagent is 1:1.5-1:5；Secondly, using anhydrous sodium acetate as catalyst, with the chloro- 1- formoxyls -3- methylols of 2- Cyclohexene carries out condensation reaction in anhydrous acetic acid acid anhydride, and reaction temperature is 25-70 DEG C, obtains seven methines with cyclohexene chlorine 3H- indoles cyanines parent dyes, structural formula are as follows：
2) under argon gas protection, using absolute methanol as solvent, n,N-diisopropylethylamine is acid binding agent, cyclohexene chlorine parent Dyestuff and the azepine derivatives progress nucleophilic substitution containing different chemical reactivity sites, parent dye and azepine derivatives Molar ratio is 1:3, reaction temperature is 40-70 DEG C.Precipitated after the completion of reaction with anhydrous ether, a large amount of solids are separated out, with dichloromethane Alkane/methanol is eluent (V dichloromethane：V methanol=20：1) combiflash companion purifying, is pressed in utilization, obtains target dye Material.
Wherein, the value of X, R1, R2, R3, R4 are as defined above.
New Heptamethine cyanines active fluoro probe provided by the invention has the Stokes shift more than 100nm, glimmering Quantum yield maximum is up to 0.4.Absorption spectrum does not have mirror symmetry relation with spectrum occurs.With traditional commerce Cy7 fluorescence Probe is compared, the nearly 130nm of maximum absorption wavelength blue shift.Due to middle position introduce chemical active radical, by chemical reaction to albumen, The biomolecule such as sugar, DNA or nano-carrier carry out fluorescent marker.Fluorescence imaging on cellular level and live body level, stablizes Property is good, and background fluorescence is low.Therefore, the present invention can be used as the biomolecule such as highly sensitive albumen, polysaccharide, DNA and organic-inorganic nano The fluorescence probe of carrier indicium, carries out cellular level and detection and imaging in live body level.
New Heptamethine cyanines active fluoro probe provided by the invention has many advantages：It is mainly reflected in three sides Face：
First, middle position azepine derivatives substitute, and since lone pair electrons have electron supplying capacity on nitrogen-atoms, swash fluorescence probe Intramolecular electron transfer occurs when sending out state, the Stokes shift of reactive monoazo dyestuffs is more than 100nm, has for practical application important Meaning.The seven methine fluorescence probe Stokes shifts currently used for fluorescence analysis are generally 25nm or so, small stoke Fluorescent dye absorption spectrum and the emission spectrum of this displacement have very big overlapping, cause self-quenching, i.e., part is launched light and inhaled by itself Receipts decline fluorescence intensity.In addition, small Stokes shift can also cause the measurement error of fluorescence analysis.Because small stoke This displacement makes excitation wavelength be got too close to fluoroscopic examination wavelength, and excitation wavelength easily causes fluoroscopic examination slit scattering dry Disturb.
Secondly, fluorescence probe of the invention introduces the chemism bases such as maleimide, disulfide bond, nitrine, acrylamide Group, can be suitable for the fluorescent marker of various biomolecules or organic-inorganic material.
Again, fluorescence probe of the invention is symmetrical structure, greatly reduces the complexity of synthesis, simplifies preparation and carries Pure technique, advantageously reduces cost.In short, such Heptamethine cyanines fluorescence probe of the present invention, integrates indoline fragrance parent nucleus Improve fluorescence intensity, rigid cyclohexene bridging improves probe steady, the substitution of middle position nitrogen substantially increases Stokes shift, activity Chemical group is easy to the advantages that polytype material fluorescent marker, can be used as the biomolecule such as highly sensitive albumen, polysaccharide, DNA and The fluorescence probe of organic-inorganic nano carrier indicium, carries out cellular level and detection and imaging in live body level.
Brief description of the drawings
Fig. 1 is the UV absorption and fluorescence emission spectrum of 2-a-a fluorescence probes.
Fig. 2 is polymer nanoparticle N-1 and N-2 fluorescence spectrum.
Fig. 3 is endocytosis of the dendritic cells (DCs) to N-1 nanoparticles.
Fig. 4 is N-1 nanoparticle living imagings.
Embodiment
With reference to specific embodiment, the present invention is further elaborated on.The experiment of actual conditions is not specified in embodiment Method, usually according to the condition described in normal condition and handbook, or according to the condition proposed by manufacturer；Used is logical With equipment, material, reagent etc., it is commercially available unless otherwise specified.
The 1 bridging parent dye of chlorine containing cyclohexene 1-a of embodiment is synthesized
According to synthetic route as shown below, the bridging parent dye 1-a of chlorine containing cyclohexene is synthesized, is comprised the following steps that：
(1) intermediate N ethyl -2,3,3- trimethyls -3H- indolines
2,3,3- trimethyl -3H- indolines (25mL, 24.8g, 156mmol) are added in the round-bottomed flask of 250mL, point Dissipate in 50mL dry toluenes, iodoethane (30g, 192mmol) anhydrous toluene solution is added dropwise, the back flow reaction under argon gas protection 12h, reaction is terminated after stopping heating.Solid product is obtained by filtration, is washed with anhydrous ether and is dried in vacuo afterwards three times, obtain pink colour Solid, yield 79%.
(2) synthesis of the chloro- 1- formoxyls -3- methylol cyclohexene of condensing agent 2-
Under condition of ice bath, 10mL phosphorus oxychloride is added dropwise dropwise in 10mL anhydrous dimethyl formamides, mixed solution is in room After the lower stirring 30min of temperature, the 10mL anhydrous dimethyl formamides solution (5.39g, 20mmol) of cyclohexanone is added dropwise dropwise, dropwise addition is opened Solution is changed into yellow at once from colourless after beginning.Reaction system reacts 3h after being heated slowly to 60 DEG C, is cooled to room temperature and pours into 200g In ice, 24h is stood in 4 DEG C of refrigerators, yellow solid is separated out, is dried in vacuo after filtering, obtains 4.5g solid products, yield 75%.
(3) the bridging parent dye of chlorine containing cyclohexene 1-a synthesis and characterization
By N- ethyls -2,3,3- trimethyl -3H- indolines quaternary amines (1.6g, 5mmol), the chloro- 1- formoxyls -3- hydroxyls of 2- Methylcyclohexene (0.43g, 2mmol) and anhydrous sodium acetate (0.41g, 5mmol) are dissolved in 15mL acetic anhydrides, and reaction system exists 1h is reacted under the conditions of 100 DEG C, stops heating and is down to room temperature.A large amount of solids are separated out after reaction solution is instilled 200mL ether, are filtered Vacuum drying obtains crude product afterwards.Combiflash companion separation (V is pressed using dichloromethane and methanol as eluent, in utilizationDichloromethane： VMethanol=10:1) 0.87g solid products, yield 65%, are obtained.1H NMR(400MHz,DMSO-d6):δ=8.36 (d, 2H, CH)；7.40(d,2H,ArH)；7.37(t,ArH,2H)；7.37(t,2H,ArH)；7.16(d,2H,ArH)；6.25(d,2H, CH)； 4.24(q,CH2,4H)；3.09(dd,2H,CH2)；2.85(dd,2H,CH2)；2.45(t,1H,CH,)；1.70(s,12H, CH3)； 1.56(m,2H,CH2)；1.44(t,CH3,6H).MS(ESI):m/z calculated:512.26；found: 512.35。
The synthesis of 2 target fluorescent probe 2-a-a of embodiment and characterization
200mg parent dyes 1-a and nucleopilic reagent N- (the 3- amino of 1.5 times of moles are added in 50mL round-bottomed flasks Propyl group) methacrylamide hydrochloride and 3.0 times of moles DIPEA, disperseed with 20mL absolute methanols, kept away under the conditions of 50 DEG C Light reaction 12h, reaction solution, which is cooled to room temperature, to be instilled in 200mL anhydrous ethers, separates out a large amount of solid precipitations, vacuum is done after filtering It is dry.Combiflash companion separation is pressed using methylene chloride/methanol as eluent, in utilization, obtains 2-a-a dyestuffs, yield 75%.
Fluorescence probe 2-a-a,1H NMR(400MHz,DMSO-d6):δ=8.73 (s, 1H, NH)；8.45(s,1H,NH)； 8.36(d,2H,CH)；7.40(d,2H,ArH)；7.37(t,ArH,2H)；7.37(t,2H,ArH)；7.16(d,2H,ArH)； 6.25 (d,2H,CH)；5.72(s,1H,CH)；5.79(s,1H,CH)；4.24(q,CH2,4H)；3.85(t,2H,CH2)；3.18 (t,2 H,CH2)；3.09(dd,2H,CH2)；2.85(dd,2H,CH2)；2.45(t,1H,CH,)；1.98(s,3H,CH3)；1.88 (q,2H, CH2)；1.70(s,12H,CH3)；1.56(m,2H,CH2)；1.44(t,CH3,6H).MS(ESI):m/z calculated:617.42；found:617.45.
The synthesis of 3 target fluorescent probe 2-a-b of embodiment and characterization
Synthetic method uses 2- (2- pyridyidithios) ethylamine hydrochloride with embodiment 2, nucleopilic reagent.
Fluorescence probe 2-a-b,1H NMR(400MHz,DMSO-d6):δ=8.73 (s, 1H, NH)；8.36(d,2H,CH)； 8.25(s,1H,CH)；7.00-8.00(m,11H,ArH)；6.25(d,2H,CH)；4.24(q,CH2,4H)；3.49(d,2H, CH2)；3.09(dd,2H,CH2)；2.85(dd,4H,CH2)；2.45(t,1H,CH,)；1.70(s,12H,CH3)；1.56(m,2H, CH2)；1.44(t,CH3,6H).MS(ESI):m/z calculated:661.34；found:661.42.
The synthesis of 4 target fluorescent probe 2-a-c of embodiment
Synthetic method uses N- (2- aminoethyls) maleimide hydrochloride with embodiment 2, nucleopilic reagent.
Fluorescence probe 2-a-c,1HNMR(400MHz,CDCl3):δ=8.36 (d, 2H, CH)；7.86(s,2H,CH)；7.40 (d, 2H,ArH)；7.37(t,ArH,2H)；7.37(t,2H,ArH)；7.16(d,2H,ArH)；6.25(d,2H,CH)；4.24 (q,CH2, 4H)；3.58(t,4H,CH2)；3.09(dd,2H,CH2)；2.85(dd,2H,CH2)；2.45(t,1H,CH,)；1.70 (s,12H, CH3)；1.56(m,2H,CH2)；1.44(t,CH3,6H).MS(ESI):m/z calculated:615.37； found:615.42。
5 fluorescence spectrometry of embodiment
Compound concentration is 1 × 10-6The 2-a-a solution (solvent be 10mM phosphate buffer solutions, pH=7.4) of M, takes dyestuff molten Liquid 3mL is placed in quartz colorimetric utensil.Absorption spectrum is measured using ultraviolet-uisible spectrophotometer and determines maximum absorption wavelength.Profit With fluorescent spectrophotometer assay fluorescence emission spectrum.As shown in Figure 1, the absorption spectrum of the new fluorescence probes of 2-a-a is relative to biography System Cy7 dyestuffs occur obvious blue shift and broaden；Second, new probe 2-a-a absorption spectrums lose mirror symmetry pass with emission spectrum System；The Stokes shift of 3rd, new probe 2-a-a reach 140nm, much larger than the Cy7 line fluorescent dyestuffs of common commercial.
The synthesis of 6 fluorescent marker polymer of embodiment
Using the polyethylene glycol that end group is three thioesters as Macromolecular chain transfer agent, reversible addition chain tra nsfer radical polymerization is utilized (RAFT), trigger methacrylic acid diisopropylaminoethyl ethyl ester (DPA) polymerization and fluorescence probe 2-a-a polymerizations, obtain fluorescent marker Polymer.Wherein, the fluorescence probe in polymer P -1 is 2-a-a, and the fluorescence probe in polymer P -2 is commercialized Cy7.Using THF as solvent, ultraviolet-uisible spectrophotometer and the spectrum of fluorescent spectrophotometer assay polymer P -1 are utilized Energy.As shown in Fig. 2, the fluorescence spectrum of contrast P-1 and 2-a-a can be seen that the method by direct radical polymerization to polymerization Thing carries out fluorescent marker, do not influence the spectrum property of fluorescence probe, it was demonstrated that 2-a-a probes are marked poly- by direct polymerization mode The feasibility of compound.
The photoluminescent property of nanoparticle prepared by embodiment 7
10mg polymer Ps -1 are weighed respectively and P-2 is dissolved in 1mL tetrahydrofurans (THF), are dripped dropwise under magnetic stirring Be added in 10mL phosphate buffer solutions (pH=7.4,10mM), volatilize THF under the conditions of lucifuge in ventilating kitchen, obtain P-1 and The corresponding nanoparticle N-1 and N-2 of P-2.Nanoparticle solution concentration is fixed as 1mg/mL, utilizes fluorescent spectrophotometer assay nanometer The fluorescence emission spectrum of grain solution.Measurement result is as shown in Figure 2.It can be seen from the figure that commercialized Cy7 fluorescence probes mark The nanoparticle of note, since Stokes shift is small, occurs serious self-quenching when being embedded in the hydrophobic inner core of N-2 nanoparticles.Phase Anti-, the 2-a-a fluorescence probes prepared in the present invention carry methacrylamide group, can by Raolical polymerizable and Monomer copolymerization, a direct step obtain the polymer of fluorescent marker；In addition, the Stokes position of prepared 2-a-a fluorescence probes Shifting is more than 120nm, solves self-quenching topic, so as to be more advantageous to carrying out fluorimetric analysis.
The dendritic cells endocytosis of embodiment 8N-1 nanoparticles
With (the FBS of hyclone containing l0%；Biochrom Ag, Germany) DMEM (Sigma-Aldrich, USA) be Basic culture solution, by cells Dendritic cells (DC2.4) with 1 × 105A/mL cell concentrations are inoculated in 24 orifice plates, be placed in 37 DEG C, 5% CO2, cultivate under the conditions of saturated humidity.After the complete adherent growth of cell, the culture medium containing N-1 nanoparticle solution is replaced. After assigning 4h, with the distribution of confocal laser scanning microscope nanoparticle in the cell.From figure 3, it can be seen that in the cell It is observed that the fluorescence signal of bright 2-a-a probes, it was demonstrated that 2-a-a fluorescent markers can be used for the fluorescence on cellular level Tracer.
9 N-1 nanoparticle living imagings of embodiment
In order to investigate the distributions of the nanoparticle N-1 of 2-a-a fluorescent markers in vivo, with the female of subcutaneous vaccination HepG2 tumours Property Balb/c nude mices tested for animal model.Experiment nude mice comes from Beijing Vital River Experimental Animals Technology Co., Ltd., Zoopery is implemented in strict accordance with National Standard of the People's Republic of China (GB/T 16886.6-1997).Specifically, choose tumour Volume about 150mm3Nude mice tail vein injection N-1 nanoparticle PBS dispersion liquids after, at specific time point, utilize living imaging The internal distribution of instrument detection nanoparticle fluorescence signal.As shown in figure 4,
After tail vein injection, significant fluorescence signal can be substantially observed in live body level, with time lengthening, swollen The enriching quantity of tumor tissue substantially increases, and shows that polymerizable 2-a-a fluorescence probes prepared by the present invention can be used in polymer nanocomposite The fluorescent marker of grain and the diagnosis of tumour.

Claims (6)

  1. A kind of 1. Heptamethine cyanines active fluoro probe Heptamethine cyanines active fluoro probe, it is characterised in that the structure of the probe General formula is as follows：
    In general formula:
    X = Ⅱ - Ⅸ；
    R1、R2Respectively： (CH2)mCH3、(CH2)nOH、(CH2CH2O)pCH3、CH2C6H5；
    R3、R4Respectively： H、SO3H、SO3Na、SO3K；
    A, b, c, d, e, f, g are respectively：2 - 8；
    N, m, p are respectively： 1-10;
    Wherein, a, b, c, d, e, f, g, n, m, p are respectively mutually independent integer.
  2. 2. the preparation method of the Heptamethine cyanines active fluoro probe described in claim 1, it is characterised in that by following step：
    1）First, starting material indoline flows back by solvent of toluene with alkylating reagent under argon gas protection, season occurs Ammoniumization is reacted, and generates alkyl-substituted indoles quaternary ammonium salt intermediate, secondly, using anhydrous sodium acetate as catalyst, with the chloro- 1- first of 2- Acyl group -3- methylols cyclohexene carries out condensation reaction in anhydrous acetic acid acid anhydride, and reaction temperature is 25-70 DEG C, obtains carrying hexamethylene Seven methine 3H- indoles cyanines parent dyes of alkene chlorine, structural formula are as follows：
    2）Under argon gas protection, using absolute methanol as solvent, n,N-diisopropylethylamine is acid binding agent, cyclohexene chlorine parent dye Nucleophilic substitution is carried out from the azepine derivatives containing different chemical reactivity sites, reaction temperature is 40-70 DEG C.React Cheng Houyong anhydrous ethers precipitate, and separate out a large amount of solids, using methylene chloride/methanol as eluent, combiflash companion is pressed in utilization Separation, obtains target product；
    Wherein, X, R1、R2、 R3、R4Value it is as defined above.
  3. 3. preparation method described in accordance with the claim 2, it is characterised in that the alkylating reagent for iodine substituted fatty hydrocarbon or Benzyl bromide a-bromotoluene.
  4. 4. preparation method described in accordance with the claim 2, it is characterised in that step 1）The indoline and alkylating reagent Molar ratio is 1:1.5-1:5.
  5. 5. preparation method described in accordance with the claim 2, it is characterised in that step 2）The parent dye and azepine derivatives Molar ratio is 1:3.
  6. 6. Heptamethine cyanines active fluoro probe described in claim 1 is used for highly sensitive albumen, polysaccharide, DNA and organic and inorganic Nano-carrier marks, and carries out cellular level and detection and imaging in live body level.
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