CN105617405A - Simple and green preparation method for monodisperse core-shell-structured C-dots@SiO2 fluorescence imaging nanoprobe - Google Patents

Abstract

The invention provides a simple and green preparation method for monodisperse core-shell-structured C-dots@SiO2 fluorescence imaging nanoprobe. The method comprises the following steps: synthesizing a carbon quantum dot (C-dots) fluorescent nano-material with a uniform size, good dispersibility and high quantum efficiency by using a microwave method; and then coating C-dots in an ethanol-water system with ethyl orthosilicate (TEOS) as a silicon source so as to form C-dots@SiO2 nanoprobe suitable for biological imaging. Due to coating of SiO2, the prepared nanoprobe is easy to separate and wash and has stable physicochemical properties and optical properties, good biocompatibility and friendliness to the environment; moreover, surface modification of the nanoprobe can be easily carried out; and the nanoprobe is applicable to other biomedical clinical application fields, e.g., targeting imaging of cancer cells.

Description

What a kind of simplicity was green prepares single dispersing nucleocapsid structure C-dotsSiO2The method of fluorescence imaging nano-probe

Technical field

The invention belongs to nano composite material and applied technical field thereof, what be specifically related to a kind of easy green prepares single dispersing nucleocapsid structure C-dotsSiO₂The method of fluorescence imaging nano-probe and its application on living imaging.

Background technology

Cancer is the disease that fatality rate is the highest in the world, and therefore, early stage detection and tracking cancerous cell have very important significance. Early-stage cancer diagnostic techniques has a lot. Such as: CT is the most frequently used diagnosis and treatment method, it is possible to present the 3D structure of tissue clearly, but most CT contrast agent of clinical practice are iodide, kidney can be caused very major injury by the excessive application of iodide. And MR imaging resolution is so not high. Owing to kidney will not be damaged by fluorescence imaging, and there is better imaging resolution on a cellular level. Therefore, fluorescence imaging is applied to imaging cancerous and will largely improve imaging effect, and the injury that organism is caused can little a lot.

Fluorescent carbon quantum dot (C-dots) has stable physicochemical properties, optical property and high-quantum efficiency, is therefore taken as very potential fluorescent bio-probes material in recent years. Simultaneously comparing against metal quantum point, C-dots has good biocompatibility, and environment does not have any pollution. (list of references: S.Zhu, J.Zhang, C.Qiao, S.Tang, Y.Li, W.Yuan, B.Li, L.Tian, F.Liu, R.Hu, H.Gao, H.Wei, H.Zhang, H.SunandB.Yang, Chem.Commun., 2011,47,6858; S.BakerandG.Baker, Angew.Chem., Int.Ed., 2010,49,6726; L.Zhou, Y.Lin, Z.Huang, J.RenandX.Qu, Chem.Commun., 2012,48,1147; B.Kong, A.Zhu, C.Ding, X.Zhao, B.LiandY.Tian, Adv.Mater., 2012,24,5844) but C-dots small-sized (less than 3nm), it is difficult to separate and washing. Therefore, in C-dots solution, excessive reaction reagent can cause non-specific adsorption to impact in ensuing bio-imaging is applied. SiO₂Shell is coated with material as one and has lot of advantages, as: good biocompatibility, physicochemical properties are stable, have good hydrophilic, it is easy to carry out finishing. Use SiO₂C-dots is coated with, it is possible to prepare the fluorescent nano probe of the good properties being easy to separation and washing. Nowadays also have and much use SiO₂The cladding fluorescent material method of preparing fluorescent nano probe, but they there are disadvantages that, as synthetic method is complicated, uses toxic pharmaceuticals in building-up process, to environment and cell there to be certain toxic action. (list of references: P.Huang, J.Lin, S.Wang, Z.Zhou, Z.Li, Z.Wang, C.Zhang, X.Yue, G.Niu, M.Yang, D.CuiandX.Chen, Biomaterials, 2013,34,4643; Z.Zhou, C.Zhang, Q.Qian, J.Ma, P.Huang, X.Zhang, L.Pan, G.Gao, H.Fu, S.Fu, H.Song, X.Zhi, J.NiandD.Cui, J.Nanobiotechnol., 2013,11,1; H.Zhang, Z.Jiao, Z.Li, M.Wu, D.PanandY.Zhang, thepreparationofanewsilicafluorescencenanosphere, China:CN101974326A, 2011-2-16)

Summary of the invention

It is an object of the invention to provide a kind of easy green method and prepare single dispersing nucleocapsid structure C-dotsSiO₂Fluorescence imaging nano-probe, uses C-dotsSiO prepared by the method₂Nano-probe has that good dispersion, physicochemical properties and optical property be stable, good biocompatibility, fluorescence intensity feature strong, eco-friendly.

Single dispersing nucleocapsid structure C-dotsSiO in the present invention₂The preparation method of nano-probe comprises the steps:

First, (1) by a certain amount of citric acid and carbamide Homogeneous phase mixing in deionized water.

(2) then above-mentioned solution is placed a period of time in 700-1000W microwave, in 60-100 DEG C of vacuum, heat 1-2h subsequently.

(3) the big particle impurities of centrifugal segregation, takes out supernatant liquid stand-by.

(4) take a certain amount of (3) solution, join in certain density sodium polyacrylate (PAAS) aqueous solution prepared, be subsequently added a certain amount of ethanol stirring 0.5-1h.

(5) with the TEOS solution that ethanol dose volume mark is 20-25%.

(6) take the solution that a certain amount of step (4) obtains, be added thereto to a certain amount of ethanol and NH₃��H₂O (2M), adds TEOS solution in (5) several times, reacts 2-3h in 40-50 DEG C of oil bath after stirring.

(7) being centrifuged the solution that step (6) obtains separating, gained solid for several times, obtains C-dotsSiO with deionized water and dehydrated alcohol alternately washing again₂��

Present invention have the advantage that

1. synthetic method is very simple, and the medicine used in building-up process does not have toxic action.

2. compared to C-dots, C-dotsSiO₂Can centrifugation easily and washing.

3. due to SiO₂The cladding of shell, the nucleocapsid structure C-dotsSiO that the present invention obtains₂Nano-probe physicochemical properties and optical property are more stable.

4. use single dispersing nucleocapsid structure C-dotsSiO prepared by the inventive method₂As bio-imaging nano-probe, there is good biocompatibility.

5.C-dots and SiO₂To environment entirely without pollution, therefore C-dotsSiO₂It it is environmental friendliness shaped material.

6.SiO₂Shell is prone to carry out finishing, the C-dotsSiO therefore generated₂It is widely used in other biological is applied.

Accompanying drawing explanation

Fig. 1, the C-dots transmission electron microscope picture prepared for the present invention;

Fig. 2, for C-dotsSiO₂Transmission electron microscope picture;

Fig. 3, the single dispersing nucleocapsid structure C-dotsSiO prepared for the present invention₂Scanning electron microscopic picture;

Fig. 4, variable concentrations C-dotsSiO₂Internal fluorescence imaging to nude mice.

Detailed description of the invention

The present invention being expanded on further below in conjunction with specific embodiment, embodiment is merely to illustrate the present invention rather than limits the scope of the invention.

Specific embodiment

Embodiment 1:

First, the Homogeneous phase mixing in 10mL deionized water by 3g citric acid and 3g carbamide. Then above-mentioned solution is placed in 700W microwave 5min, in 60 DEG C of vacuum, heats 1h subsequently. Under 3000rpm rotating speed, centrifugal 20min removes big particle impurities, takes out supernatant liquid stand-by.

Take the 350 above-mentioned C-dots solution of �� L, join 10mLPAAS aqueous solution (2gL^-1) in, it is subsequently added 50mL ethanol, stirs 30min. Take the solution that above-mentioned steps obtains, be added thereto to 180mL ethanol and 70mLNH₃��H₂O (2M), adds 20%TEOS solution (preparing with ethanol) several times after stirring, react 2h when 40 DEG C. (after first, adding 100 �� LTEOS, 40min, add 100 �� L, remaining 800 �� LTEOS add 200 �� L every 20min) last, being centrifuged the mixed solution obtained separating, gained solid for several times, obtains C-dotsSiO with deionized water and dehydrated alcohol alternately washing again₂��

Embodiment 2:

First, the Homogeneous phase mixing in 15mL deionized water by 6g citric acid and 8g carbamide. Then above-mentioned solution is placed in 850W microwave 20min, in 80 DEG C of vacuum, heats 1.5h subsequently. Under 3000rpm rotating speed, centrifugal 20min removes big particle impurities, takes out supernatant liquid stand-by.

Take the 500 above-mentioned C-dots solution of �� L, join 20mLPAAS aqueous solution (2gL^-1) in, it is subsequently added 100mL ethanol, stirs 45min. Take the solution that above-mentioned steps obtains, be added thereto to 100mL ethanol and 50mLNH₃��H₂O (2M), adds 20%TEOS solution (preparing body with ethanol) several times after stirring, react 2.5h when 45 DEG C. (after first, adding 250 �� LTEOS, 40min, add 250 �� L, remaining 2000 �� LTEOS add 300 �� L every 20min) last, being centrifuged the mixed solution obtained separating, gained solid for several times, obtains C-dotsSiO with deionized water and dehydrated alcohol alternately washing again₂��

Embodiment 3:

First, the Homogeneous phase mixing in 20mL deionized water by 8g citric acid and 10g carbamide. Then above-mentioned solution is placed in 1000W microwave 10min, in 100 DEG C of vacuum, heats 2h subsequently. Under 6000rpm rotating speed, centrifugal 20min removes big particle impurities, takes out supernatant liquid stand-by.

Take the 500 above-mentioned C-dots solution of �� L, join 20mLPAAS aqueous solution (2gL^-1) in, it is subsequently added 100mL ethanol, stirs 60min. Take the solution that above-mentioned steps obtains, be added thereto to 150mL ethanol and 100mLNH₃��H₂O (2M), adds 25%TEOS solution (preparing with ethanol) several times after stirring, react 3h when 50 DEG C. (after first, adding 400 �� LTEOS, 40min, add 400 �� L, remaining 2800 �� LTEOS add 700 �� L every 20min) last, being centrifuged the mixed solution obtained separating, gained solid for several times, obtains C-dotsSiO with deionized water and dehydrated alcohol alternately washing again₂��

The single dispersing nucleocapsid structure C-dotsSiO of above-mentioned synthesis₂Nano composite material is optically active substance, can be used for internal fluorescence imaging.

Internal fluorescence imaging step is: according to every kilogram to the amount anesthetized mice of anaesthetic 10mL (pentobarbital sodium 0.7%). Then, the C-dotsSiO of the 200 �� L variable concentrations will prepared with PBS (pH=7.4)₂Solution in-situ injection, in mouse back, then carries out fluorescence imaging.

Claims (5)

1. convenient for cleaning prepare single dispersing nucleocapsid structure C-dotsSiO for one kind₂The method of fluorescence imaging nano-probe, comprises the steps: the preparation of fluorescent carbon quantum dot (C-dots), uses SiO₂Cladding C-dots prepares C-dotsSiO₂Fluorescence imaging nano-probe.

2. method according to claim 1, it is characterised in that: SiO₂The fluorescent material of cladding is C-dots, SiO₂With C-dots to environment and organism nonhazardous effect.

3. method according to claim 1, cladding process as template, is then coated with upper SiO with sodium polyacrylate (PAAS)₂��

4. method according to claim 1, cladding process carries out under strongly alkaline conditions, and PH is more than 9.

5. method according to claim 1, the cladding used time is very short, for 2-3h.