CN101348713A - Magnetic composite nano-microsphere capable of emitting fluorescence and preparation method thereof - Google Patents

Abstract

The invention discloses a magnetic composite nano microsphere capable of emitting fluorescent light and a preparation method thereof, which belong to the inorganic nanophase material field. The preparation method comprises the following steps: firstly, the sol-gel method or the reversed phase microemulsion method is adopted for performing base catalysis on alkyl silicate, and a layer of silicon dioxide is uniformly coated on the surface of inorganic magnetic nano particles; secondly, an amino silane coupling agent is adopted for performing surface finish on silicone dioxide; thirdly, semiconductor quantum dots are absorbed on the surface of the silicone dioxide through the electrostatic action between an amino group and a carboxyl group on the surface of the amino silane coupling agent; and fourthly, the magnetic composite nano microsphere with a core-shell structure capable of emitting the fluorescent light is obtained. The preparation method has easy obtained raw materials and low cost. The composite nano microsphere obtained has good stability, controllable grain size, narrow distribution, and has good magnetic responsivity and performance of being capable of emitting the fluorescent light, and the two performances are controllable. The magnetic composite nano microsphere and the preparation method can be applied in the fields of biomedicine, bioengineering, etc. including immunoassay, biological labeling and separation, cell imaging and so on.

Description

Magnetic composite nano microsphere of energy emitting fluorescence and preparation method thereof

Technical field

The invention belongs to field of inorganic nano material, be specifically related to a kind of fluorescent, magnetic, multi-functional composite nano-microsphere and preparation method thereof.

Background technology

Quantum dot (QDs) be meant radius less than or nanocrystalline near the exciton Bohr radius, be that a class is different from the type material that body differs from molecule, atomic properties again.Quantum dot is because its unique quantum size effect and surface effects, thereby has special and good visible region fluorescent emission character.Alivisatos and Nie in 1998 on first phase Science reported first quantum dot replace conventional dyestuff to be used for biomarker and cell imaging as fluorescent probe, indicate that the work that the research quantum dot is applied in the biomedical sector begins.Since then, quantum dot has caused vast chemistry, biology and medical research personnel's strong interest with its excellent specific property.Compare with traditional fluorescence organic dye, the quantum dot emission spectral position only depends on its particle diameter, cover from the ultraviolet to the region of ultra-red, thereby can be simply by changing the quantum dot that particle diameter obtains having different glow colors.Utilize its excitation spectrum continuously, the narrow and symmetric characteristic in emmission spectrum peak, cross interference in the time of can significantly reducing the hyperchannel fluoroscopic examination, select for use a kind of excitation wavelength just can realize multi-color marking and the joint inspection of many indexs, and its fluorescence spectrum is stable, influenced by solvent, pH value, temperature etc., can realize the ultra-long time Real-time and Dynamic Detection.Because of its excellent specific property, quantum dot has represented great application prospect in fields such as biological chemistry, molecular biology, cytobiology, genomics, proteomics, drug screening and interaction of biomacromolecules.

Magnetic nano-particle has special magnetic property, is widely used at biomedical sector.Target biological molecules or target cell are adsorbed on the biomagnetism nanoparticle by functional group, and the outside magnetic field effect can realize separating of protein purification, cell and nucleic acid etc. down.Behind the magnetic medicine carrier medicine carrying, the outside magnetic field effect is orientable to move to the target place and discharges medicine, thereby reaches the purpose of targeted therapy.After magnetic particle imported to the tumour body, under extraneous action of alternating magnetic field, absorb hertzian wave and be converted into heat energy, after reaching required treatment temperature, tumour cell is killed, reach the effect of tumor thermotherapy.

With quantum dot and magnetic nano-particle combination, obtain a kind of quantum dot fluorescence effect of both having had, have the composite nano materials of magnetic particle magnetic property again, have very big potential using value.At present quantum dot and magnetic nano-particle compound mainly contains two kinds of methods: (1) is embedded in both in the body material simultaneously.As magnetic particle and quantum dot are embedded in (Yi D K, Selvan T, Lee S S, et al.J.Am.Chem.Soc., 2005,127:4990 in the silica nanosphere simultaneously; Kim J, Lee J E, L J, et al.J.Am.Chem.Soc., 2006,128,688-689; The Chinese patent publication number is CN1698582), magnetic particle and quantum dot are embedded in (the Chinese patent publication number is CN1539913) in the polymer microsphere simultaneously.(2) quantum dot is adsorbed on the magnetic particle surface.Wang etc. directly are adsorbed on the CdSe/ZnS quantum dot γ-Fe of polymer overmold by sulfydryl₂O₃The surface, and the fluoroscopic examination of using it for cancer cells separate with magnetic (Wang D, He J, RosenzweigN, et al.Nano Lett., 2004,4:409).Hong etc. adopt layer-by-layer at Fe₃O₄Particle surface adsorbs quantum dot after adsorbing certain number of plies polyelectrolyte again, perhaps alternately adsorb polyelectrolyte and quantum dot, obtain fluorescence magnetic composite nano materials (Hong X, Li J, Wang M, et al.Chem.Mater., 2004,16:4022), and applied for relevant patent (the Chinese patent publication number is CN1524925).Publication number is that 1831079 Chinese patent utilizes the aminopropyl silane coupling agent to modify Fe₃O₄Particle by the electrostatic interaction between amino and the carboxyl, directly is assembled in Fe with quantum dot again₃O₄Particle surface, the shortcoming of this method are that the quantum dot adsorptive capacity is difficult to control, and two kinds of particles are owing to directly contact, and its interaction can cause fluorescence intensity sharply to descend.

Among the present invention, we have proposed, and a kind of raw material is easy to get, the simple method for preparing the fluorescent, magnetic, multi-functional composite nano-microsphere, the Nano microsphere regular shape, and particle diameter, fluorescence and magnetic are controlled.

Summary of the invention

In the prior art with quantum dot and Fe₃O₄Particle directly adsorbs, and has the easily shortcoming of loss of adsorptive capacity control difficulty, fluorescence intensity

The objective of the invention is to propose a kind of fluorescent, magnetic, multi-functional composite nano-microsphere and preparation method thereof.

The fluorescent, magnetic, multi-functional composite nano-microsphere that the present invention proposes, be a kind of be that nuclear, silicon-dioxide are shell with the inorganic magnetic nanoparticle, and at the core-shell type composite nano-microsphere of its surface adsorption quantum point.Because this composite nano-microsphere has magnetic kernel, therefore have magnetic responsiveness, simultaneously because the silica shell surface adsorption has quantum dot, thereby under the UV, visible light optical excitation, have fluorescence property.By changing silicon dioxide thickness (between 5-50nm), the composite nano-microsphere particle diameter is changed in 20-200nm, magnetic particle and quantum dot quantity ratio are 1: 10-1: between 100.In addition, with the increase of silicon dioxide thickness, the composite nano-microsphere particle diameter increases, its surface-area increases, thus the more quantum dots of absorption, and distance increases between magnetic nano-particle and the quantum dot, can reduce the interference of magnetic particle greatly, strengthen the fluorescence intensity of composite nano-microsphere fluorescence.

The preparation of the above-mentioned fluorescent, magnetic, multi-functional composite nano-microsphere that the present invention proposes, at first be magnetic nano-particle to be carried out surface modification with organic acid, adopt sol-gel method or reverse microemulsion process at the adjustable silicon-dioxide of magnetic nano-particle coated with uniform one layer thickness then, adopt amino silicane coupling agent that silica sphere is modified again, obtaining a kind of is nuclear with the magnetic nano-particle, silicon-dioxide is shell, amino composite nanometer particle is rich on the surface of shell, quantum dot last and surface band carboxyl mixes, electrostatic interaction by between the amino and electronegative carboxyl of positively charged is assembled in silica sphere with quantum dot.

The thickness of above-mentioned silica shell can be simply by changing tetraalkyl orthosilicate, and promptly the consumption of silicon-dioxide presoma is controlled.By the thickness of change silicon-dioxide and the kind and the particle diameter of quantum dot, can obtain having the fluorescence magnetic composite nano-microsphere of different magnetic responsivenesses, different fluorescence intensity and glow color, promptly can be used for multicolor fluorescence detects, can test product separation, orientation and enrichment again, be applicable to fields such as biological chemistry, molecular biology and cytobiology.

Concrete preparation process is as follows:

(1) preparation of inorganic magnetic nanometer particle colloid: the inorganic magnetic nanoparticle is dispersed in the organic acid soln, use ammoniacal liquor or NaOH solution to regulate the pH value as between the 4-8, magnetic separates after continuing to stir 4-5h, use washed with de-ionized water 2-4 time then, be dispersed in the deionized water at last, obtain stable magnetic nano-particle colloid, magnetic particles is 0.5-5.0wt%.

(2) to prepare silicon-dioxide be shell, the magnetic particle composite nanometer particle for nuclear to sol-gel method: the magnetic particle colloid of step 1) preparation is dispersed in the ethanol/water solution, adds ammoniacal liquor and tetraalkyl orthosilicate then, 20-40 ℃ continues to stir 4-12h down.In the whole ethanol/water solution reaction system, magnetic particle is 0.01-0.1wt%, and ethanol is 60-80wt%, and water is 15-35wt%, and ammoniacal liquor is 1.5-3.5wt%, and all the other are tetraalkyl orthosilicate.

(3) prepared by reverse microemulsion method silicon-dioxide is shell, the composite nanometer particle of magnetic particle for examining: the magnetic particle colloid of step (1) preparation is dispersed in the water in oil reverse microemulsion system, add ammoniacal liquor and tetraalkyl orthosilicate then, 20-40 ℃ continues to stir 8-16h down.In the whole reaction system, magnetic particle is 0.01-0.1wt%, and oil phase is 45-55wt%, and water is 9-14wt%, and tensio-active agent is 30-40wt%, and ammoniacal liquor is 0.3-0.5wt%, and all the other are tetraalkyl orthosilicate.

(4) surface amination of silica shell: with step 2) or 3) the nucleocapsid structure composite nanometer particle of preparation is dispersed in the ethanolic soln, adds small amount of deionized water, amino silicane coupling agent successively, 20-80 ℃ continues down to stir 4-12h.In the whole reaction system, step 2) or 3) composite nanometer particle of preparation is 0.1-1.0wt%, ethanol is 92-96wt%, water is 2-6wt%, all the other are amino silicane coupling agent.

(5) preparation of fluorescence magnetic composite nano-microsphere: with the semiconductor-quantum-point of the composite nanometer particle of the surface amination of step 4) preparation and surface band carboxyl with 1: 20-1: 100 mixed in molar ratio, regulating pH value be 6-8, the lasting 2-8h that adsorbs under the room temperature.

The fluorescence magnetic composite nano-microsphere of above method preparation can separate and use deionized water wash under the magnet effect.

Among the present invention, used magnetic nano-particle is the Fe of particle diameter between 5-50nm₃O₄, γ-Fe₂O₃, Mn_1-xZn_xFe₂O₄(wherein X is between 0.1-0.6), CoFe₂O₄Or NiFe₂O₄A kind of.

Among the present invention, used organic acid is that citric acid, two hydration citric acids, tartrate, oxalic acid etc. contain a plurality of carboxyls or have the organic acid of hydroxyl a kind of simultaneously.

Among the present invention, used tetraalkyl orthosilicate is a kind of in methyl silicate, the tetraethoxy.

Among the present invention, used amino silicane coupling agent is a kind of in 3-TSL 8330,3-aminopropyltriethoxywerene werene, N-(β-aminoethyl)-γ-An Bingjisanjiayangjiguiwan.

Among the present invention, used semiconductor-quantum-point be particle diameter between 3-8nm, CdSe, CdTe, CdS or the ZnS of surface band carboxyl one or more, wherein above-mentioned quantum dot is through the Thiovanic acid finishing.

Description of drawings

Fig. 1 is embodiment 3 synthetic CdSe/SiO₂/ Fe₃O₄TEM figure;

Fig. 2 is embodiment 3 synthetic CdSe/SiO₂/ Fe₃O₄Magnetic hysteresis loop;

Fig. 3 is embodiment 3 synthetic CdSe/SiO₂/ Fe₃O₄Fluorogram.

Embodiment

The testing tool of Fig. 1 is the H-800 transmission electron microscope of Hitachi company; The testing tool of Fig. 2 is the LH-3 vibrating sample magnetometer of Nanjing Univ. Instrument Factory; The testing tool of Fig. 3 is the 970CRT spectrophotofluorometer of Shanghai Precision Scientific Apparatus Co., Ltd.

Embodiment 1:

(1) in the 500ml three-necked bottle, adding the 5.0g particle diameter is the ferroferric oxide powder of 10nm, add the 200ml deionized water, dispersed with stirring added 2.1g two hydration citric acids after 2 hours under the 500rpm rotating speed, and using the ammonia soln adjusting pH value of 1mol/l is 6.0, continue to stir after 5 hours and separate with magnet, and with washed with de-ionized water 3-5 time, at last Z 250 is dispersed in the 200ml deionized water, make the magnetic fluid that solid content is 2.0wt%.

(2) in a 500ml three-necked bottle, add 40ml deionized water, 160ml dehydrated alcohol successively, add the magnetic fluid that 2g prepares, the strong aqua and the 0.2ml tetraethoxy that under the 500rpm rotating speed, add 4ml 25wt% then, 25 ℃ continue down to stir after 8 hours, magnetic is used washed with de-ionized water 3 times after separating successively, and dehydrated alcohol cleans 3 times.

(3) in a 150ml round bottom beaker, the magnetic-particle of above-mentioned coated with silica all is dispersed in the 50ml dehydrated alcohol, add the 2ml deionized water again, treat to add when system temperature rises to 50 ℃ 0.1ml 3-TSL 8330, the 500rpm rotating speed continues down to stir that magnetic separates after 12 hours, and uses dehydrated alcohol and washed with de-ionized water respectively 2 times.

(4) in a 100ml round bottom beaker, silica magnetic particle after the above-mentioned amination all is dispersed in the 10ml deionized water, adding aqueous phase is the CdSe quantum dot of stablizer preparation with the Thiovanic acid, HCl and NaOH solution adjusting pH value with 1mol/l are 6.5, stir magnetic separation after 2 hours, and, obtain the CdSe/SiO that particle diameter is 50nm with washed with de-ionizedwater 5 times₂/ Fe₃O₄The fluorescence magnetic composite nano-microsphere.

Embodiment 2:

(1) in the 500ml three-necked bottle, add the ferroferric oxide powder of 5.0g through deionized water wash, add the 200ml deionized water, dispersed with stirring added 2.1g two hydration citric acids after 2 hours under the 500rpm rotating speed, and using the ammonia soln adjusting pH value of 1mol/l is 6.0, continue to stir after 5 hours and separate with magnet, and with washed with de-ionized water 3-5 time, at last Z 250 is dispersed in the 200ml deionized water, make the magnetic fluid that solid content is 2.0wt%.

(2) in a 500ml three-necked bottle, add the magnetic fluid that 80ml hexanaphthene, 28ml Triton X-100 (Triton X-100), 20ml hexanol, 2g prepare successively, the strong aqua that adds 12ml deionized water and 0.6ml 25% then, 25 ℃ and 500rpm rotating speed stir down and add the 0.2ml tetraethoxy after 20 minutes, continue to stir after 12 hours, after separating, cleans 2 times with acetone successively magnetic, washed with de-ionized water 3 times, and dehydrated alcohol cleans 3 times.

(3) in a 150ml round bottom beaker, the magnetic particle of above-mentioned coated with silica all is dispersed in the 50ml dehydrated alcohol, add the 2ml deionized water again, treat to add when system temperature rises to 50 ℃ 0.1ml 3-TSL 8330, the 500rpm rotating speed continues down to stir that magnetic separates after 12 hours, and uses dehydrated alcohol and washed with de-ionized water respectively 2 times.

(4) in a 100ml round bottom beaker, silica magnetic particle after the above-mentioned amination all is dispersed in the 10ml deionized water, adding aqueous phase is the CdSe quantum dot of stablizer preparation with the Thiovanic acid, HCl and NaOH solution adjusting pH value with 1mol/l are 6.5, stir magnetic separation after 2 hours, and, obtain the CdSe/SiO that particle diameter is 50nm with washed with de-ionizedwater 5 times₂/ Fe₃O₄The fluorescence magnetic composite nano-microsphere.

Embodiment 3:

Change the consumption of the tetraethoxy of the step (2) of embodiment (1) into 0.8ml, all the other conditionally complete unanimities obtain the CdSe/SiO that particle diameter is 100nm₂/ Fe₃O₄The fluorescence magnetic composite nano-microsphere.Its TEM, magnetic hysteresis loop figure and fluorescence pattern are respectively as Fig. 1, Fig. 2 and shown in Figure 3.

Embodiment 4:

Change the consumption of the tetraethoxy of the step (2) of embodiment (2) into 0.8ml, all the other conditionally complete unanimities obtain the CdSe/SiO that particle diameter is 100nm₂/ Fe₃O₄The fluorescence magnetic composite nano-microsphere.

Embodiment 5:

Fe with the step (1) of embodiment (1)₃O₄Consumption replace with Mn_0.7Zn_0.3Fe₂O₄, all the other conditionally complete unanimities obtain the CdSe/SiO that particle diameter is 50nm₂/ Mn_0.7Zn_0.3Fe₂O₄The fluorescence magnetic composite nano-microsphere.

Embodiment 6:

Fe with the step (1) of embodiment (1)₃O₄Consumption replace with Mn_0.7Zn_0.3Fe₂O₄, the consumption of the tetraethoxy of step (2) changes 0.8ml into, and all the other conditionally complete unanimities obtain the CdSe/SiO that particle diameter is 100nm₂/ Mn_0.7Zn_0.3Fe₂O₄The fluorescence magnetic composite nano-microsphere.

Embodiment 7:

The CdSe of the step (4) of embodiment (1) is replaced with CdS, and all the other conditionally complete unanimities obtain the CdS/SiO that particle diameter is 50nm₂/ Fe₃O₄The fluorescence magnetic composite nano-microsphere.

Embodiment 8:

The tetraethoxy of the step (2) of embodiment (1) is replaced with methyl silicate, and all the other conditionally complete unanimities obtain the CdSe/SiO that particle diameter is 50nm₂/ Fe₃O₄The fluorescence magnetic composite nano-microsphere.

Embodiment 9:

The 3-TSL 8330 of the step (3) of embodiment (1) is replaced with N-(β-aminoethyl)-γ-An Bingjisanjiayangjiguiwan, and all the other conditionally complete unanimities obtain the CdSe/SiO that particle diameter is 50nm₂/ Fe₃O₄The fluorescence magnetic composite nano-microsphere.

Claims (8)

1. the magnetic composite nano microsphere of an energy emitting fluorescence is characterized in that with single or multiple inorganic magnetic nanoparticles be kernel, is shell with the silicon-dioxide of surface amination, and semiconductor-quantum-point evenly is adsorbed on the surface of silica shell; The thickness of silicon dioxide layer is between 5-50nm, and the composite nano-microsphere particle diameter is between the 20-200nm scope; Magnetic particle and quantum dot quantity ratio are 1: 10-1: between 100.

2. composite nano-microsphere according to claim 1 is characterized in that described inorganic magnetic nanoparticle is the Fe of particle diameter between 5-50nm₃O₄, γ-Fe₂O₃, Mn_1-xZn_xFe₂O₄(wherein X is between 0.1-0.6), CoFe₂O₄Or NiFe₂O₄In a kind of.

3. composite nano-microsphere according to claim 1, it is characterized in that described quantum dot be particle diameter between 3-8nm, and among CdSe, CdTe, CdS or the ZnS of surface band carboxyl one or more, wherein above-mentioned quantum dot is through the Thiovanic acid finishing.

4. preparation is according to the method for any described composite nano-microsphere among the claim 1-3, it is characterized in that: at first magnetic nano-particle is carried out surface modification with organic acid, adopt sol-gel method or reverse microemulsion process at the adjustable silicon-dioxide of magnetic nano-particle coated with uniform one layer thickness then, again by silicon-dioxide being carried out surface modification with amino silane coupling agent, make the silica sphere amination, by the electrostatic interaction between the carboxyl on amino and fluorescence quantum surface, quantum dot is adsorbed on silica sphere at last.

5. method according to claim 4 is characterized in that concrete steps comprise:

(1) preparation of inorganic magnetic nanometer particle colloid:

The inorganic magnetic nanoparticle is dispersed in the organic acid soln, use ammoniacal liquor or NaOH solution to regulate the pH value between 4-8, magnetic separates after continuing to stir 4-5h, use washed with de-ionized water 2-4 time then, be dispersed in the deionized water at last, obtain stable magnetic nano-particle colloid, magnetic particles is between 0.5-5.0wt%;

(2) to prepare silicon-dioxide be shell, the magnetic particle composite nanometer particle for nuclear to sol-gel method:

The magnetic particle colloid of step (1) preparation is dispersed in the ethanol/water solution, adds ammoniacal liquor and tetraalkyl orthosilicate then, 20-40 ℃ continues to stir 4-12h down; In the whole reaction system, magnetic particle is 0.01-0.1wt%, and ethanol is 60-80wt%, and water is 15-35wt%, and ammoniacal liquor is 1.5-3.5wt%, and all the other are tetraalkyl orthosilicate;

(3) surface amination of silica shell:

The nucleocapsid structure composite nanometer particle of step (2) or (3) preparation is dispersed in the ethanolic soln, adds small amount of deionized water, amino silicane coupling agent successively, 20-80 ℃ continues to stir 4-12h down; In the whole reaction system, step 2) or 3) composite nanometer particle of preparation is between 0.1-1.0wt%, ethanol is between 92-96wt%, water is between 2-6wt%, all the other are amino silicane coupling agent;

(4) preparation of fluorescence magnetic composite nano-microsphere:

With the semiconductor-quantum-point of the composite nanometer particle of the surface amination of step (4) preparation and surface band carboxyl with 1: 20-1: 100 mixed in molar ratio, regulate pH value between 6-8, continue to adsorb 2-8h under the room temperature.

Method according to claim 4, it is characterized in that it is shell, the composite nanometer particle of magnetic particle for examining that step (2) can also adopt reverse microemulsion process to replace sol-gel method to prepare silicon-dioxide, specifically: the magnetic particle colloid of step (1) preparation is dispersed in the water in oil reverse microemulsion system, add ammoniacal liquor and tetraalkyl orthosilicate then, 20-40 ℃ continues to stir 8-16h down; In the whole reaction system, magnetic particle is 0.01-0.1wt%, and oil phase is 45-55wt%, and water is 9-14wt%, and tensio-active agent is 30-40wt%, and ammoniacal liquor is 0.3-0.5wt%, and all the other are tetraalkyl orthosilicate; Wherein, oil phase is a hexanaphthene, and water is a deionized water, and tensio-active agent is the mixture of Triton X-100 and hexanol.

7. method according to claim 5 is characterized in that described organic acid is the organic acid that contains a plurality of carboxyls simultaneously or have hydroxyl; Specifically comprise citric acid, two hydration citric acids, tartrate, oxalic acid.

8. method according to claim 5 is characterized in that described tetraalkyl orthosilicate is a kind of in methyl silicate, the tetraethoxy.

9. method according to claim 5 is characterized in that described amino silicane coupling agent is a kind of in 3-TSL 8330,3-aminopropyltriethoxywerene werene, N-(β-aminoethyl)-γ-An Bingjisanjiayangjiguiwan.

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