CN102961762A - Method for wrapping fluorescent protein by nanosilicon dioxide - Google Patents
Method for wrapping fluorescent protein by nanosilicon dioxide Download PDFInfo
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- CN102961762A CN102961762A CN2012104429483A CN201210442948A CN102961762A CN 102961762 A CN102961762 A CN 102961762A CN 2012104429483 A CN2012104429483 A CN 2012104429483A CN 201210442948 A CN201210442948 A CN 201210442948A CN 102961762 A CN102961762 A CN 102961762A
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- fluorescin
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- aminopropyl triethoxysilane
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- silicon dioxide
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Abstract
The invention relates to a method for wrapping fluorescent protein by nanosilicon dioxide, and belongs to the field of inorganic nano materials and biological medicines. The method mainly comprises the following steps of: carrying out covalent linkage between a certain amount of 3-aminopropyl triethoxy silane and protein; and then catalyzing tetraethoxysilane and the covalent linkage product of the 3-aminopropyl triethoxy silane and protein to hydrolyze through basic amino acid, thus obtaining fluorescent nanosilicon dioxide particles wrapped with near-infrared, infrared and green fluorescent proteins. The method is simple and easy to carry out; the prepared nanometer particles are extremely high in stability, dispersibility and biosecurity, and are hardly subjected to enzymolysis; and the method can be successfully applied to living imaging of small animals.
Description
Technical field
The present invention relates to a kind of method of utilizing nano silicon parcel fluorescin, belong to nano inorganic material and biochemical field.
Background technology
Nano silicon is a kind of inorganic nano material of nontoxic, good biocompatibility, and its surface is easy to modify and modification, is widely used in the biomedical sectors such as biomarker, nano-probe, medicine-carried system.Utilize nano silicon parcel specific molecular to prepare special material and use very extensively, as utilize the nano silicon packaging medicine, utilize nano silicon parcel fluorescent material to improve quantum yield etc., but its potential source biomolecule toxicity is to evade the question.The present invention's Silica-coated fluorescin of reporting for work first has excellent biocompatibility and dispersibility and is successfully applied to the small animal living body imaging.
Summary of the invention
According to the defective that prior art exists, the purpose of this invention is to provide a kind of method of utilizing nano silicon parcel fluorescin, and be applicable to other protein
,Have following process and step:
A. 3-aminopropyl triethoxysilane and fluorescin are covalently bound: get the fluorescin of 1-100 mg and the 3-aminopropyl triethoxysilane of 50-1000 ul, reaction namely gets product through G 25 desalinations and 1000-10000 g ultrafiltration in the presence of 10-500 mg 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 10-500 mg N-maloyl imines;
B. wrap up the preparation of the nano silicon of near-infrared fluorescent albumen: fully stir evenly in the basic amino acid adding deionized water with 50-500 mg, add above-mentioned 0.1-1 ml 3-aminopropyl triethoxysilane and the covalently bound product of fluorescin, add again 10-100 ul tetraethoxysilane and cyclohexane extraction, after stirring at room reacts completely, the impurity such as aminoacid are removed in 3500g ultrafiltration washing for 3-5 time, can obtain comprising the nanometer silicon dioxide particle of fluorescin.
Compared with prior art, the present invention has following outstanding advantage:
The present invention utilizes a kind of nanometer silicon dioxide particle of simple method preparation parcel fluorescin, fluorescin is wrapped in nano silicon inside, improved the stability of fluorescin by parcel, has better heat stability and pH stability, and have and be difficult for being difficult for by good performances such as cancellation by enzymolysis and fluorescence, that has greatly expanded albumen utilizes scope and field.The nanometer silicon dioxide particle that comprises fluorescin of gained is dispersed in the normal saline, by the toy tail vein injection, can success in the small animal living body imaging system carry out the video picture of toy optics.
Description of drawings
Fig. 1 is transmission electron microscope (TEM) the photo figure that gained of the present invention comprises the nanometer silicon dioxide particle of near-infrared fluorescent albumen.
Fig. 2 is dynamic light scattering (DLS) figure that gained of the present invention comprises the nanometer silicon dioxide particle of near-infrared fluorescent albumen.
Fig. 3 is that gained of the present invention is the fluorescence spectrum figure with the nanometer silicon dioxide particle that comprises near-infrared fluorescent albumen under the isoconcentration and near-infrared fluorescent albumen.
Fig. 4 is nanometer silicon dioxide particle and the near-infrared fluorescent albumen correlation curve figure in mouse liver S9 system that gained of the present invention comprises near-infrared fluorescent albumen.
Fig. 5 is nanometer silicon dioxide particle and the near-infrared fluorescent proteolysis correlation curve figure that gained of the present invention comprises near-infrared fluorescent albumen.
Fig. 6 is the small animal living body image that gained of the present invention directly wraps up the nanometer silicon dioxide particle mouse tail vein injection of near-infrared fluorescent albumen.
The specific embodiment
Below in conjunction with accompanying drawing, after specific embodiments of the invention are discussed in.
A. 3-aminopropyl triethoxysilane and near-infrared fluorescent albumen are covalently bound: get a certain amount of near-infrared fluorescent albumen (eqFP650), be dissolved in 14.6 milliliters of pH and be 7.5 phosphate buffer, add 200 milligrams of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 250 milligrams of N-maloyl imines, add again 400 microlitre 3-aminopropyl triethoxysilanes, magnetic agitation 24 hours namely gets product through desalination and ultrafiltration;
B. wrap up the preparation of the nano silicon of near-infrared fluorescent albumen (eqFP650): get 75mg lysine and add in the 15ml deionized water and fully stir evenly, add the above-mentioned 3-aminopropyl triethoxysilane of 2.1ml and the covalently bound product of fluorescin, add again 18ul tetraethoxysilane and 12ul cyclohexane extraction, room temperature vigorous stirring reaction 12 hours is progressively adding 18ul tetraethoxysilane and 12ul cyclohexane extraction stirring at room 12 hours to complete reaction.The impurity such as aminoacid are removed in ultrafiltration washing (3500g/minute about) for several times, can obtain comprising the nanometer silicon dioxide particle of near-infrared fluorescent albumen.
C. the nanometer silicon dioxide particle that comprises near-infrared fluorescent albumen (eqFP650) with gained is dispersed in the normal saline, passes through mouse tail vein injection.Can success in the small animal living body imaging system carry out the video picture of toy optics.
A. 3-aminopropyl triethoxysilane and near-infrared fluorescent albumen are covalently bound: get a certain amount of near-infrared fluorescent albumen (eqFP650), be dissolved in 14.6 milliliters of pH and be 7.5 phosphate buffer, add 200 milligrams of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 250 milligrams of N-maloyl imines, add again 400 microlitre 3-aminopropyl triethoxysilanes, magnetic agitation 24 hours namely gets product through desalination and ultrafiltration;
B. wrap up the preparation of the nano silicon of mNeptune fluorescin: get 100mg lysine and add in the 15ml deionized water and fully stir evenly, add the above-mentioned 3-aminopropyl triethoxysilane of 1.1ml and the covalently bound product of fluorescin, add again 18ul tetraethoxysilane and 12ul cyclohexane extraction, room temperature vigorous stirring reaction 12 hours is progressively adding 18ul tetraethoxysilane and 12ul cyclohexane extraction stirring at room 12 hours to complete reaction.The impurity such as aminoacid are removed in ultrafiltration washing (4000 g/minute about) for several times, can obtain comprising the nanometer silicon dioxide particle of near-infrared fluorescent albumen.
C. the nanometer silicon dioxide particle that comprises the mNeptune fluorescin with gained is dispersed in the normal saline, passes through mouse tail vein injection.Can success in the small animal living body imaging system carry out the video picture of toy optics.
Embodiment 3
Near-infrared fluorescent albumen among the embodiment 1 can be changed into other Infrared fluorescence albumen (Infrared Fluorescent Proteins), other operational approach are with reference to embodiment 1, and the nanometer titanium dioxide silicon materials that still can obtain wrapping up Infrared fluorescence albumen are applied in the living imaging.
A. the prepared nanometer silicon dioxide particle that comprises fluorescin has excellent dispersibility.With the nanoparticulate dispersed of preparation at phosphate buffer, normal saline, cell culture fluid leaves standstill in the Ox blood serum still can be dispersed in three months in the solution and precipitation occurs.
B. the prepared nanometer silicon dioxide particle that comprises fluorescin has significant lifting at more simple fluorescin aspect stability and the fluorescence intensity.Prepared fluorescent nano particle is at antidegenerative agents, proteolysis resistant, and there is significant lifting anti-Mouse Liver S9 aspect than fluorescin, and under same concentrations during its fluorescence intensity 1.5 times of simple fluorescin.
C. the prepared nanometer silicon dioxide particle that comprises fluorescin has excellent biocompatibility and biological safety.The nano-particle of prepared parcel fluorescin has excellent biocompatibility with respect to traditional quantum dot fluorescence probe that contains heavy metal, can not produce potential potential safety hazard to organism.
D. the fluorescin of nano silicon parcel can success is applied to the animal horizontal imaging.The nanometer silicon dioxide particle that comprises fluorescin of gained is dispersed in the normal saline, passes through mouse tail vein injection.Can success in the small animal living body imaging system carry out the video picture of toy optics.
Claims (1)
1. method of utilizing nano silicon parcel fluorescin is characterized in that having following step:
A. 3-aminopropyl triethoxysilane and fluorescin are covalently bound: get the fluorescin of 1-100 mg and the 3-aminopropyl triethoxysilane of 50-1000 ul, reaction namely gets product through G 25 desalinations and 1000-10000 g ultrafiltration in the presence of 10-500 mg 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 10-500 mg N-maloyl imines;
B. wrap up the preparation of the nano silicon of fluorescin: fully stir evenly in the basic amino acid adding deionized water with 50-500 mg, add above-mentioned 3-aminopropyl triethoxysilane and the covalently bound product 0.1-3 of fluorescin ml, add again 10-100 ul tetraethoxysilane and cyclohexane extraction, after stirring at room reacts completely, the impurity such as aminoacid are removed in 3500g ultrafiltration washing for 3-5 time, can obtain comprising the nanometer silicon dioxide particle of fluorescin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333250A (en) * | 2013-06-24 | 2013-10-02 | 上海大学 | Method for preparing nano fluorescent probe with high bio-safety |
| CN110229806A (en) * | 2019-05-28 | 2019-09-13 | 上海大学 | Nano silica wraps up trypsase and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101137579A (en) * | 2005-03-10 | 2008-03-05 | 独立行政法人科学技术振兴机构 | Regularly arrayed nanoparticle silica, and its manufacturing method |
| CN101792480A (en) * | 2010-03-17 | 2010-08-04 | 上海大学 | Method for packing histidine-tagged protein with nano silica |
| CN101805407A (en) * | 2010-03-17 | 2010-08-18 | 上海大学 | Method for coating protein by nanometer silicon dioxide |
| CN102072891A (en) * | 2009-11-20 | 2011-05-25 | 中国科学院化学研究所 | Metal-modified photonic crystal biological detection film as well as preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101137579A (en) * | 2005-03-10 | 2008-03-05 | 独立行政法人科学技术振兴机构 | Regularly arrayed nanoparticle silica, and its manufacturing method |
| CN102072891A (en) * | 2009-11-20 | 2011-05-25 | 中国科学院化学研究所 | Metal-modified photonic crystal biological detection film as well as preparation method and application thereof |
| CN101792480A (en) * | 2010-03-17 | 2010-08-04 | 上海大学 | Method for packing histidine-tagged protein with nano silica |
| CN101805407A (en) * | 2010-03-17 | 2010-08-18 | 上海大学 | Method for coating protein by nanometer silicon dioxide |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333250A (en) * | 2013-06-24 | 2013-10-02 | 上海大学 | Method for preparing nano fluorescent probe with high bio-safety |
| CN110229806A (en) * | 2019-05-28 | 2019-09-13 | 上海大学 | Nano silica wraps up trypsase and preparation method thereof |
| CN110229806B (en) * | 2019-05-28 | 2023-05-16 | 上海大学 | Nanometer silicon dioxide coated trypsin and preparation method thereof |
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Application publication date: 20130313 |