CN102660255A - Magnetic fluorescent nanoparticle with biological activity and method for preparing magnetic fluorescent nanoparticle - Google Patents
Magnetic fluorescent nanoparticle with biological activity and method for preparing magnetic fluorescent nanoparticle Download PDFInfo
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Abstract
The invention discloses a magnetic fluorescent nanoparticle with biological activity and a method for preparing the magnetic fluorescent nanoparticle, belongs to the technical field of preparation of biological imaging materials, and particularly belongs to the field of magnetic imaging and fluorescent imaging. The nanoparticle prepared by the method is provided with a core-shell structure, a core of the nanoparticle is made of ferroferric oxide so that the nanoparticle has a fine magnetic performance, a shell of the nanoparticle is made of silicon dioxide, functions in protecting the ferroferric oxide and provides a platform for surface modification, biological molecules with carboxyl or amino and fluorescent compounds with amino or carboxylic acid groups are led into the surface of the nanoparticle by means of reaction of the amino and the carboxyl, and the biological molecules have biological activity, so that the nanoparticle has fine biological compatibility and excellent optical performance. The nanoparticle prepared by the method simultaneously has excellent magnetic performance, biological activity and fluorescent performance, and accordingly has a wide application prospect in the field of biomedicine including life sciences, genetics, pharmaceutics, cell imaging and the like.
Description
Technical field
The invention belongs to the bio-imaging technical field of material, be specially magnetic imaging and fluorescence imaging, particularly magnetic fluorescent nanometer particle of a kind of biologically active and preparation method thereof.
Background technology
Magnetic microsphere is formed by magnetic nano-particle and macromolecular scaffold material prepn.Macromolecular material wherein comprises PS, silane, Vilaterm, ROHM, starch, VISOSE, gelatin, BSA, Mierocrystalline cellulose etc.There is natural materials that synthetic materials is also arranged, can uses separately also can share and make framework material.These framework material stable in properties, intensity are higher, have no side effect, so can be described as immune magnetic microsphere.No matter be that experimental study or clinical application all show, immune magnetic microsphere is just being brought into play enormous function in biomedicine and drug release.Magnetic nanoparticle after surface modified is widely used in cellular segregation, immunoassay, enzyme with aspects such as deciding, nuclear magnetic resonance, target drug-carrying, tumour magnetic mediation thermotherapies.
In recent years; In field of nanometer material technology; Research is exactly to use the detection that various fluorescent probes carry out biologically active substance the most widely, and these fluorescent probes should have following characteristics: light stability is preferably arranged, be difficult for by photodissociation or bleaching, little to the influence of the function of organism own, have and good excite, the organism of being surveyed is had good imaging and mark effect with fluorescence efficiency.Simultaneously; Through modification to fluorescent particles, introduce the different surface structure, make particle can be used as the carrier of gene and medicine to a certain extent; Do not destroy simultaneously the fluorescent characteristic of particle itself, make us to follow the tracks of the transfection of gene and the dispose procedure of medicine.
The nuclear of magnetic microsphere is Z 250.The main character of Z 250 is superparamagnetism.When the grain diameter of magnetic nano-particle during less than critical size, the polarity of magnet presents randomness, does not have hysteresis, remove foreign field after, its remanent magnetism will disappear very soon, residual magnetization is almost nil, ultra paramagnetic effect that Here it is.The generation of superparamagnetism and the particle diameter of magnetic Nano material have very big relation, and its critical size of different magnetic Nano materials is different, and the material of subcritical size is called super paramagnetic material.The great advantage of this material is exactly after externally-applied magnetic field is removed, and does not have residual magnetization.This magnetism characteristic makes to have extensive use in the numerous areas such as magnetic Nano material separates at nuclear magnetic resonance, magnetic, magnetic target medicine transports.
In numerous fluorescent chemicalses, condensed-nuclei aromatics is one type of arene compound that intense fluorescence is arranged, and has big conjugated pi system.Verivate has excellent chemistry, light and heat chemicalstability; Light from the visible region to the infrared region there is very strong absorption; Be one type of ME material that performance is special, be widely used aspect laserable material, biological fluorescent labeling molecule, liquid crystal display material, electroluminescent device, photoreceptor and the solar cell.Wherein, electron conjugated degree and molecule same flatness in Nai 、 perylene and the terylene equimolecular are very big, therefore have very high fluorescence quantum yield.Because this compounds fluorescence is very strong, therefore the property research to them all is the problem of Showed Very Brisk with using exploration all the time.
Summary of the invention
The objective of the invention is in order to prepare simultaneously at the nanoparticle that all has good characteristic aspect magnetic, fluorescence property and the biological activity three.
The nanoparticle that the present invention makes is a nucleocapsid structure, and wherein the nuclear of particle is Z 250, for particle provides good magnetic behavior; The shell of particle is a silicon-dioxide, has played the effect of protection Z 250, for surface-treated platform is provided simultaneously; Reaction through amino and carboxyl has carboxyl or an amino biomolecules and the fluorescent chemicals that has amino or hydroxy-acid group to what particle surface was introduced biologically active then, makes this nanoparticle have the optical property of excellent biological compatibility and excellence.
The preparation process of the magnetic fluorescent nanometer particle of biologically active of the present invention is:
(1) with the FeCl of 0.1-3.0g
36H
2O and 0.1-6.0g sodium-acetate are dissolved in 20-150ml terepthaloyl moietie or Diethylene Glycol solvent, perhaps in terepthaloyl moietie and the Diethylene Glycol mixed solvent, obtain yellow mixing solutions after stirring is also ultrasonic; Being positioned over pressure then is 1.01 * 10
5Pa-1 * 10
6In the autoclave of Pa, 180-220 ℃ of reaction 1-24h; Reaction is cooled to room temperature after finishing, and product is separated with magnet, uses ethanol, water, acetone then successively, and 25-100 ℃ of vacuum-drying 1-24h obtains magnetic particle (MNP);
(2) under ultra-sonic dispersion and churned mechanically condition, magnetic particle and 0.01-5ml tetraethoxy (TEOS) that 5mg-0.5g step (1) is obtained are dispersed in the 10-150ml volume ratio in the ethanol and water mixed solvent of (1: 10)-(10: 1); In system, add the ethanolic soln of 10.1-55ml ammoniacal liquor behind the dispersion 5-30min, massfraction is the ammoniacal liquor 0.1-5ml of 3%-15% in the ethanolic soln of described ammoniacal liquor, ethanol 10-50ml; Room temperature reaction 1-24h after product separates with magnet, uses ethanol, water, acetone then respectively, 25-100 ℃ of vacuum-drying 1-24h;
(3) the ultrasonic and mechanical dispersed with stirring of product that 5mg-0.5g step (2) is obtained is in the toluene of 150mL; Add 0.1-5ml γ-chloropropyl triethoxysilane (APTES) then, use the magnet separated product behind the room temperature reaction 1-24h, use ethanol, water, acetone respectively, 20-100 ℃ of vacuum-drying 1-24h;
(4) 10mg-0.2g is had carboxyl or amino biomolecules and 1mg-0.1g catalyst dissolution in the phosphate buffer soln of 10-50ml PH=6-6.3; Add the product that 5mg-0.1g step (3) obtains then; Ultrasonic reaction 10-30min between-10 ℃ to 10 ℃, stirring at room is reacted 1-72h, magnet separated product then; Phosphate buffer soln with ethanol, water, acetone, PH=6-6.3 cleans 20-100 ℃ of vacuum-drying 1-24h respectively;
(5) optical dye and the 1mg-0.1g catalyst dissolution that 1mg-0.1g are had amino or carboxyl are in the phosphate buffer soln of 10-50ml PH=6-6.3; Add the product that 1mg-0.5g step (4) obtains then, ultrasonic agitation reaction 5-30min, stirring reaction 1-72h under the room temperature then in ice bath; The magnet separated product cleans with the phosphate buffer soln of ethanol, water, acetone, PH=6-6.3 respectively, and 20-100 ℃ of vacuum-drying 1-24h obtains the magnetic fluorescent nanometer particle of biologically active.
The biomolecules that has carboxyl or amino described in the step (4) is CMS (CMCS), CMC 99.5, CMS or polyamino acid.
The described structural formula that has the optical dye of amino or carboxyl is:
Wherein, R
1The ionic groups that has sulfonic group and quaternary ammonium salt for C1-C10; R
2The group that has carboxyl or amino for C1-C20.
Described catalyzer is that mass ratio is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and the N-hydroxy-succinamide (NHS) of (1: 10)-(10: 1).
The magnetic fluorescent nanometer particle of described biologically active is a kernel with the Z 250 of particle diameter 6-200nm, is shell with silicon-dioxide, and particle surface is biomolecules and fluorophor.
Compare with traditional particle; The nanoparticle of the present invention's preparation has had both the good characteristic of magnetic, biological activity and fluorescence property three aspects, makes it have broad application prospects at biomedicine fields such as life science, genetics, pharmacology and cell imagings.
Description of drawings
The reaction principle figure of step in Fig. 1 claim 1 (1)-(4).
The reaction principle figure of step in Fig. 2 claim 1 (5).
The ultraviolet of the magnetic fluorescent nanometer particle of the biologically active that Fig. 3 embodiment 1 makes-fluorescence spectrum spectrogram.
The projection electron microscope photo of the magnetic fluorescent nanometer particle of the biologically active that Fig. 4 embodiment 1 makes.
Specific embodiments
Embodiment 1
(1) with the FeCl of 1g
36H
2It is in 1: 1 the terepthaloyl moietie and Diethylene Glycol mixed solvent that O and 3g sodium-acetate are dissolved in the 60ml volume ratio, stir and ultrasonic after obtain yellow mixing solutions; Being positioned over pressure then is 1.01 * 10
6In the autoclave of Pa, 210 ℃ of reaction 10h; Reaction is cooled to room temperature after finishing, and product is separated with magnet, cleans respectively 5 times with ethanol, water, acetone successively then, and 25 ℃ of vacuum-drying 12h obtain the pulverous magnetic particle 0.83g of black solid, and productive rate is 96.3%;
(2) under ultra-sonic dispersion and churned mechanically condition, it is in 5: 1 the ethanol and water mixed solvent that magnetic particle that 0.2g step (1) is obtained and 1mL tetraethoxy are dispersed in the 24ml volume ratio; In system, add the ethanolic soln of 25ml ammoniacal liquor behind the dispersion 30min, wherein massfraction is 15% ammoniacal liquor 5ml, ethanol 20ml; Room temperature reaction 24h after product separates with magnet, uses ethanol, water, acetone then respectively, 60 ℃ of vacuum-drying 5h;
(3) the ultrasonic and mechanical dispersed with stirring of product that 0.05g step (2) is obtained is in the toluene of 150mL; Add 5mL γ-chloropropyl triethoxysilane then, use the magnet separated product behind the room temperature reaction 24h, use ethanol, water, acetone respectively 5 times, 25 ℃ of vacuum-drying 10h;
(4) with the 20mg CMS; 20mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 24mg N-hydroxy-succinamide are dissolved in the phosphate buffer soln of 40ml PH=6, add the product that 40mg step (3) obtains then, 0 ℃ of ultrasonic reaction 10min; Stirring at room is reacted 24h then; The magnet separated product, the phosphate buffer soln with ethanol, water, acetone, PH=6 cleans 25 ℃ of vacuum-drying 24h respectively;
(5) 1mg is had the amino perhaps optical dye DYE-SO of carboxyl
3H-COOH and 10mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 12mg N-hydroxy-succinamide are dissolved in the phosphate buffer soln of 40mlPH=6; Add the product that 10mg step (4) obtains then, ultrasonic agitation reaction 10min, stirring reaction 24h under the room temperature then in ice bath; The magnet separated product cleans with the phosphate buffer soln of ethanol, water, acetone, PH=6 respectively, and 25 ℃ of vacuum-drying 10h obtain the magnetic fluorescent nanometer particle of biologically active.
The described optical dye DYE-SO that has amino or carboxyl
3The structural formula of H-COOH is:
Embodiment 2
(1) with the FeCl of 1g
36H
2It is in 1: 5 the terepthaloyl moietie and Diethylene Glycol mixed solvent that O and 3g sodium-acetate are dissolved in the 60ml volume ratio, stir and ultrasonic after obtain yellow mixing solutions; Being positioned over pressure then is 1.01 * 10
6In the autoclave of Pa, 210 ℃ of reaction 10h; Reaction is cooled to room temperature after finishing, and product is separated with magnet, cleans respectively 5 times with ethanol, water, acetone successively then, and 25 ℃ of vacuum-drying 12h obtain the pulverous magnetic particle 0.83g of black solid, and productive rate is 96.3%;
(2) under ultra-sonic dispersion and churned mechanically condition, it is in 5: 1 the ethanol and water mixed solvent that magnetic particle that 0.2g step (1) is obtained and 1mL tetraethoxy are dispersed in the 24ml volume ratio; In system, add the ethanolic soln of 25ml ammoniacal liquor behind the dispersion 30min, wherein massfraction is 15% ammoniacal liquor 5ml, ethanol 20ml; Room temperature reaction 24h after product separates with magnet, uses ethanol, water, acetone then respectively, 60 ℃ of vacuum-drying 5h;
(3) the ultrasonic and mechanical dispersed with stirring of product that 0.05g step (2) is obtained is in the toluene of 150mL; Add 5mL γ-chloropropyl triethoxysilane then, use the magnet separated product behind the room temperature reaction 24h, use ethanol, water, acetone respectively 5 times, 25 ℃ of vacuum-drying 10h;
(4) with the 20mg CMS; 20mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 24mg N-hydroxy-succinamide are dissolved in the phosphate buffer soln of 40ml PH=6, add the product that 40mg step (3) obtains then, 0 ℃ of ultrasonic reaction 10min; Stirring at room is reacted 24h then; The magnet separated product, the phosphate buffer soln with ethanol, water, acetone, PH=6 cleans 25 ℃ of vacuum-drying 24h respectively;
(5) 1mg is had the amino perhaps optical dye DYE-N of carboxyl
+(CH
3)
3-COOH and 10mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 12mg N-hydroxy-succinamide are dissolved in the phosphate buffer soln of 40ml PH=6; Add the product that 10mg step (4) obtains then, ultrasonic agitation reaction 10min, stirring reaction 24h under the room temperature then in ice bath; The magnet separated product cleans with the phosphate buffer soln of ethanol, water, acetone, PH=6 respectively, and 25 ℃ of vacuum-drying 10h obtain the magnetic fluorescent nanometer particle of biologically active.
The described optical dye DYE-N that has amino or carboxyl
+(CH
3)
3The structural formula of-COOH is:
Embodiment 3
(1) with the FeCl of 1g
36H
2It is in 1: 3 the terepthaloyl moietie and Diethylene Glycol mixed solvent that O and 3g sodium-acetate are dissolved in the 60ml volume ratio, stir and ultrasonic after obtain yellow mixing solutions; Being positioned over pressure then is 1.01 * 10
6In the autoclave of Pa, 210 ℃ of reaction 10h; Reaction is cooled to room temperature after finishing, and product is separated with magnet, cleans respectively 5 times with ethanol, water, acetone successively then, and 25 ℃ of vacuum-drying 12h obtain the pulverous magnetic particle of black solid;
(2) under ultra-sonic dispersion and churned mechanically condition, it is in 5: 1 the ethanol and water mixed solvent that magnetic particle that 0.2g step (1) is obtained and 1mL tetraethoxy are dispersed in the 24ml volume ratio; In system, add the ethanolic soln of 25ml ammoniacal liquor behind the dispersion 30min, wherein massfraction is 15% ammoniacal liquor 5ml, ethanol 20ml; Room temperature reaction 24h after product separates with magnet, uses ethanol, water, acetone then respectively, 60 ℃ of vacuum-drying 5h;
(3) the ultrasonic and mechanical dispersed with stirring of product that 0.05g step (2) is obtained is in the toluene of 150mL; Add 5mL γ-chloropropyl triethoxysilane then, use the magnet separated product behind the room temperature reaction 24h, use ethanol, water, acetone respectively 5 times, 25 ℃ of vacuum-drying 10h;
(4) with the 20mg polymerization degree be 10 polylysine; 20mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 24mg N-hydroxy-succinamide are dissolved in the phosphate buffer soln of 40ml PH=6, add the product that 40mg step (3) obtains then, 0 ℃ of ultrasonic reaction 10min; Stirring at room is reacted 24h then; The magnet separated product, the phosphate buffer soln with ethanol, water, acetone, PH=6 cleans 25 ℃ of vacuum-drying 24h respectively;
(5) 1mg is had the amino perhaps optical dye DYE-SO of carboxyl
3H-COOH and 10mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 12mg N-hydroxy-succinamide are dissolved in the phosphate buffer soln of 40mlPH=6; Add the product that 10mg step (4) obtains then, ultrasonic agitation reaction 10min, stirring reaction 24h under the room temperature then in ice bath; The magnet separated product cleans with the phosphate buffer soln of ethanol, water, acetone, PH=6 respectively, and 25 ℃ of vacuum-drying 10h obtain the magnetic fluorescent nanometer particle of biologically active.
The described optical dye DYE-SO that has amino or carboxyl
3The structural formula of H-COOH is:
Claims (5)
1. the preparation method of the magnetic fluorescent nanometer particle of a biologically active is characterized in that, its concrete preparation process is:
(1) with the FeCl of 0.1-3.0g
36H
2O and 0.1-6.0g sodium-acetate are dissolved in 20-150ml terepthaloyl moietie or Diethylene Glycol solvent, perhaps in terepthaloyl moietie and the Diethylene Glycol mixed solvent, obtain yellow mixing solutions after stirring is also ultrasonic; Being positioned over pressure then is 1.01 * 10
5Pa-1 * 10
6In the autoclave of Pa, 180-220 ℃ of reaction 1-24h; Reaction is cooled to room temperature after finishing, and product is separated with magnet, uses ethanol, water, acetone then successively, and 25-100 ℃ of vacuum-drying 1-24h obtains magnetic particle;
(2) under ultra-sonic dispersion and churned mechanically condition, magnetic particle and 0.01-5ml tetraethoxy that 5mg-0.5g step (1) is obtained are dispersed in the 10-150ml volume ratio in the ethanol and water mixed solvent of (1: 10)-(10: 1); In system, add the ethanolic soln of 10.1-55ml ammoniacal liquor behind the dispersion 5-30min, massfraction is the ammoniacal liquor 0.1-5ml of 3%-15% in the ethanolic soln of described ammoniacal liquor, ethanol 10-50ml; Room temperature reaction 1-24h after product separates with magnet, uses ethanol, water, acetone then respectively, 25-100 ℃ of vacuum-drying 1-24h;
(3) the ultrasonic and mechanical dispersed with stirring of product that 5mg-0.5g step (2) is obtained is in the toluene of 150mL; Add 0.1-5ml γ-chloropropyl triethoxysilane then, use the magnet separated product behind the room temperature reaction 1-24h, use ethanol, water, acetone respectively, 20-100 ℃ of vacuum-drying 1-24h;
(4) 10mg-0.2g is had carboxyl or amino biomolecules and 1mg-0.1g catalyst dissolution in the phosphate buffer soln of 10-50ml PH=6-6.3; Add the product that 5mg-0.1g step (3) obtains then; Ultrasonic reaction 10-30min between-10 ℃ to 10 ℃, stirring at room is reacted 1-72h, magnet separated product then; Phosphate buffer soln with ethanol, water, acetone, PH=6-6.3 cleans 20-100 ℃ of vacuum-drying 1-24h respectively;
(5) optical dye and the 1mg-0.1g catalyst dissolution that 1mg-0.1g are had amino or carboxyl are in the phosphate buffer soln of 10-50ml PH=6-6.3; Add the product that 1mg-0.5g step (4) obtains then, ultrasonic agitation reaction 5-30min, stirring reaction 1-72h under the room temperature then in ice bath; The magnet separated product cleans with the phosphate buffer soln of ethanol, water, acetone, PH=6-6.3 respectively, and 20-100 ℃ of vacuum-drying 1-24h obtains the magnetic fluorescent nanometer particle of biologically active.
2. the preparation method of the magnetic fluorescent nanometer particle of a kind of biologically active according to claim 1; It is characterized in that the biomolecules that has carboxyl or amino described in the step (4) is CMS, CMC 99.5, CMS or polyamino acid.
3. the preparation method of the magnetic fluorescent nanometer particle of a kind of biologically active according to claim 1 is characterized in that, the described structural formula that has the optical dye of amino or carboxyl is:
Wherein, R
1The ionic groups that has sulfonic group and quaternary ammonium salt for C1-C10; R
2The group that has carboxyl or amino for C1-C20.
4. the preparation method of the magnetic fluorescent nanometer particle of a kind of biologically active according to claim 1; It is characterized in that described catalyzer is that mass ratio is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy-succinamide of (1: 10)-(10: 1).
5. the preparation method of the magnetic fluorescent nanometer particle of a kind of biologically active according to claim 1; It is characterized in that; The magnetic fluorescent nanometer particle of described biologically active is a kernel with the Z 250 of particle diameter 6-200nm; With silicon-dioxide is shell, and particle surface is biomolecules and fluorophor.
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