CN1183999C - Nm-class core-and-shell particles - Google Patents
Nm-class core-and-shell particles Download PDFInfo
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- CN1183999C CN1183999C CN 00113681 CN00113681A CN1183999C CN 1183999 C CN1183999 C CN 1183999C CN 00113681 CN00113681 CN 00113681 CN 00113681 A CN00113681 A CN 00113681A CN 1183999 C CN1183999 C CN 1183999C
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention discloses a nanometer particle applied to the high-sensitivity identification of chemistry, biology and physics. Nanometer matter with one of characteristics of light, electricity and magnetism is used as an inner core. The inner core is contained in a package casing prepared through a chemical or physical package method and has a chemical combination function. The inner core can be a quantum dot with special fluorescent performance, a magnetic nanometer particle with a magnetic guiding characteristic, a semiconductor or a metal nanometer particle, a dye microcapsule and other inorganic or organic nanometer accumulation bodies. A package medium can also be arranged between the inner core and a casing, and the surface of the inner core and a hydrophilic or oleophilic medium contacting the casing can be evenly packed.
Description
The present invention relates to nanoparticles, further be meant the nano particle of identification such as the high sensitivity that is applied to chemistry or biology, physics, medical science.
Nano particle is meant diameter at the molecule of 1 nanometer to the hundreds of nanometer range, because material presented under this yardstick ins and outs and function develop the research of nano-substance and application rapidly.
The objective of the invention is, develop nm-class core-and-shell particles, be applied to identifying such as chemistry, biology, physics, medical science and have high-sensitive recognition reaction, its application will make correlation technique obtain breakthrough.
Technical scheme of the present invention is, the structure of described nm-class core-and-shell particles is, it is a kernel with the nano-substance with one of light, electricity, magnetic characteristic, and this kernel is included in and adopts chemistry or physical package package method to be prepared from and have in the package shell of chemical bond function.
Among the present invention, described nano inner core can be the quantum dot (as the CdSe quantum dot) that special fluorescence property is arranged, the magnetic nano particle that the magnetic steering characteristic is arranged, semiconductor nanoparticle, metal nanoparticle, dyestuff micro-capsule and other inorganic or organic nano accumulation body.Between described kernel and shell the parcel medium can also be arranged, described parcel medium can adopt the inorganic or organic compound that can evenly wrap up the nano inner core surface and blindly date with shell.
Owing to have the nano particle kernel of particular characteristic and these materials of large scale relatively have more excellent characteristic; and the excellent specific property of these nano particle kernels of bare state is subjected to the interference of extraneous factor and variation easily; and these nano inner cores are difficult to directly with linking in conjunction with having the chemical cross-linking agent unit of chemistry with the material of bio-identification effect; the proposition of nm-class core-and-shell particles of the present invention; because of shell to the protective effect of kernel and its chemical bond function, solved above-mentioned technical problem breakthroughly.
Nm-class core-and-shell particles of the present invention can be realized a series of biology, chemistry, physical identification application on this basis as the basic function body.
For example, in package shell, forming by the chemical cross-linking agent unit can be in conjunction with the active group with chemistry or bio-identification, combination, the chemical cross-linking agent unit is to adopt chemical cross-linking agent and package shell to react, one end of its molecule is connected to package shell and the other end has active group, as-NH
2,-OH ,-COO etc. can combine with the material of bio-identification and combination with having chemistry, also promptly have chemistry and combine with package shell by active group with the material of bio-identification and combination.These materials with chemistry and bio-identification and combination can be a series of biological substances such as enzyme, antigen-antibody, DNA, RNA, protein, polysaccharide or organic polymer, organic molecule and inorganic compound etc.
Nm-class core-and-shell particles of the present invention just becomes the nano particle with corresponding recognition function after the chemical cross-linking agent unit has connected described material with chemistry or bio-identification and combination.Its generation principle with high sensitivity recognition reaction is, when the entrained recognition material of described nano particle and measured object are had an effect, the correlation properties generation marked change of the nano inner core with excellent specific property will be made, this variation fully can be reflected by the respective detection means, promptly realize highly sensitive identification.
A kind of physical identification of nm-class core-and-shell particles of the present invention is used and is, utilize magnetisable material to do nano inner core, and make particle carry certain drug molecule (material) of treatment disease, and at the disease sites complementary field, the nano particle that then carries medicine is concentrated to disease sites under the effect of magnetic field, can give full play to the effect of medicine thus, improve result of treatment.
As known from the above, the present invention has developed described nm-class core-and-shell particles, and it is applied to chemistry, biology, physical identification process, has the high sensitivity recognition reaction, can be widely used in numerous areas such as chemistry, biology, medical treatment.
Embodiment 1: immunofluorescence nano particle identification SmlgG+B lymphocyte
(1) preparation of fluorescent nano particle.In the system that contains surfactant Qu Latong XTritonX-100, thiacyclohexane and hexanol, be decentralized photo with an amount of water, add water-soluble dye Ru (bpy)
36H
2O, prepare water in oil microemulsion, with the formed silica copolymerization of ammonia-catalyzed ethyl orthosilicate (TEOS) hydrolysis, above mixture continues to stir and stops after 24 hours reacting, make the fluorescence silicon nano particles that embed fluorescent dye, particle with acetone separation, centrifugal, is successively washed repeatedly to supernatant with absolute ethyl alcohol and deionization and do not had fluorescence, and suspended particulate is standby in water.
(2) nano grain surface is handled and the antibody connection.Particle is suspended in 2MNa
2CO
3In the solution, supersonic oscillations 15 minutes make hydroxyl on the particle surface activation zone, add cyanogen bromide (CNBr) solution of acetonitrile dissolving, stir 10 minutes under the room temperature.The particle of handling is used the PBS washed twice of frozen water and PH7.6 respectively, discrete particles, the goat anti-human igg antibody's solution that adds phosphate buffer (PBS) dilution of 100uLPH7.6,4 ℃ of following continuous stirring add the glycine reactant 30 minutes of 0.03M after 24 hours, product is suspended in the PBS solution of PH7.6 after washing for several times with PBS, promptly obtains to possess the nano particle of immune identification action.
(3) experimental result
(3.1) feature of nano particle
(A) size of particle and shape
Fig. 1 is the transmission electron microscope photo of fluorescent nano particle, the result shows that to utilize the fluorescent nano particle of microemulsion that the little bag method of Water-In-Oil forms and silica copolymerization gained spherical in shape, the size basically identical, diameter is at 60 ± 5nm, and the size of particle can change by other parameter when that changes water and surfactant.Described microemulsion be by water, oil, surfactant and cosurfactant four components such as (as alcohols) with the spontaneous transparent or semitransparent stabilising system that forms of proper proportion, it divides two types: (1) water-in-oil type: interior be water mutually, the foreign minister is oily; (2) oil-in-water type: interior is oil mutually, and the foreign minister is a water.
Fig. 2 is fluorescent nano particle (1ml, 1.5ml, the 2ml in the aqueous solution of being suspended in that gets different volumes respectively, 2.5ml, 3ml), the fluorescence spectrum under EX463nm excites, fluorescence intensity and volume are good linear relationship, illustrate also that thus the size of nano particle is more even.
(B) the fluorescence spectrum characteristic of fluorescent grain
A. fluorescent material Ru (bpy)
36H
2After O was embedded in and makes fluorescent nano particle in the nano particle, orchid had been taken place and has moved (moving to 594nm from the 609nm orchid) in fluorescence spectrum, sees Fig. 3 and Fig. 4, and this may be that energy level increases after being wrapped because of dye molecule.
B. the antibody Covalent Immobilization is in the surface of fluorescent nano particle, and the optical property of fluorescent nano particle is not caused variation, excites down at EX463nm, and maximum emission wavelength still is 594nm, sees Fig. 5.
(C) oxygen is to the influence of fluorescent nano particle luminous intensity
Ru (bpy)
36H
2O is a kind of fluorescent material to oxygen sensitive, and oxygen has strong putting out not have effect to its fluorescence intensity, and Fig. 6 is Ru (bpy)
36H
2The fluorescence intensity of the aqueous solution of O and feed pure N
2After fluorescence intensity, the result shows the feeding high-purity N
2After, because of the reduction fluorescence intensity of solution value of partial pressure of oxygen sharply raises; But for the formed luminescence from silicon nano particle of Water-In-Oil, extraneous partial pressure of oxygen reduces the fluorescence intensity that does not make particle (see figure 7) takes place to change significantly.Therefore, Ru (bpy)
36H
2After O is packed the nano particle that becomes to have fluorescence, can avoid in the external environment some potential factors to cause extinguishing of fluorescence and bleach, thereby improve the stability of nano-particle fluorescence intensity.
(3.2) the immunofluorescence nano particle of sessile antibody is to the lymphocytic identification of SmIgG+B
Fig. 8-the 11st, bone-marrow-derived lymphocyte and the cytological map of nano particle cultivation after 1 hour that is fixed with the goat anti-human igg, Fig. 8, Fig. 9 are the ordinary optical microscope observed result, Figure 10, Figure 11 are the fluorescence microscope result, and fluorescigenic cell is SmIgG in Figure 10, Figure 11
+Bone-marrow-derived lymphocyte; Figure 12 and Figure 13 are the results of control experiment, promptly bone-marrow-derived lymphocyte with not fixedly goat anti-human igg antibody's nanometer fruit grain cultivate cytological map after 1 hour, be respectively that ordinary optical microscope is observed and the result of fluorescence microscope, cell does not all have fluorescence.
Because of the fluorescent nano particle that the goat anti-human igg antibody is arranged is surely cultivated with the bone-marrow-derived lymphocyte of living, the corresponding Immunoglobulin IgG generation antigen-antibody binding reaction of the goat anti-human igg antibody on fluorescent nano particle surface and bone-marrow-derived lymphocyte surface, make the be fixed fluorescent nano particle sign of antibody of part lymphocyte, under fluorescence microscope, be viewed as the cell of red fluorescence, be SmIgG
+Bone-marrow-derived lymphocyte fluoresces and not fluorescent bone-marrow-derived lymphocyte number (removing dead cell) by statistics, draws in normal person's peripheral blood SmIgG
+The percentage of bone-marrow-derived lymphocyte is about 8.33%, and this result and FITC sign antibody or SPA detect SmIgG in normal person's peripheral blood
+Bone-marrow-derived lymphocyte is close, but it shows SmIgG
+The antibody height that the signal to noise ratio of bone-marrow-derived lymphocyte fluorescence intensity indicates than FITC, because when this fluorescent nano particle of preparation, fluorescent material Ru (bpy)
36H
2O is a saturated aqueous solution, can wrap in the nano particle of an about 60nm by many fluorescence molecules, and therefore under antibody molecule and the man-to-man situation of nano particle, antibody molecule has connected many fluorescence molecules; And adopt fluorescein isothiocynate (FITC) sign antibody molecule, FITC is to combine with the epsilon-amino of lysine residue on the antibody, at most can only be in conjunction with 8-15 fluorescein molecule for an antibody molecule.
In control experiment, the surface of fluorescent grain does not have sessile antibody, and cell can not interact with it and antigen-antibody binding reaction takes place and be indicated, thereby all cells do not have fluorescence, sees Figure 13.
More than experiment repeats repeatedly, all draws identical experimental result, therefore can utilize this fluorescent nano particle sign goat anti-human igg antibody to realize SmIgG
+The identification of bone-marrow-derived lymphocyte.
More than experiment has shown that the nano silicon particles of the embedding fluorescence molecule for preparing by Water-In-Oil has the advantage that many semiconductor crystal materials do not have: the program that nano particle is made is simple, directly prepares the spherical fluorescent nano particle of tool silica surface, big or small basically identical by the netted silica copolymerization of Water-In-Oil formation microemulsion and teos hydrolysis formation; The good hydrophilic property of particle, energy and bio-compatible have realized the film surface immunity of single bone-marrow-derived lymphocyte is also needed the identification of protein I gG; The fluorescent nano particle of tool silica surface is easy to functionalization, can directly utilize cyanogen bromide to modify and connect antibody molecule; The fluorescence intensity of fluorescent nano particle is stable, and the variation of external environment partial pressure of oxygen can not cause fluorescent material Ru (bpy) in the particle
36H
2O fluorescence intensity obvious variation, thus some latencies (external environment of depending on for existence as the oxidation reaction in the cell system and cell) extinguishing or bleach avoided to fluorescence intensity.
Utilize the advantage of this fluorescent nano particle to realize in this experiment to SmIgG in normal person's peripheral blood
+The identification of bone-marrow-derived lymphocyte, we also can be according to different experiment needs, other has the biomolecule (as antibody, DNA, differential protein etc.) of special identification function at the surface bond of fluorescent nano particle, realizes special Recognition of Biomolecular or mensuration such as the surface marker (as antigen, acceptor etc.) of individual cells or DNA; Because its high sensitivity is added size and the shape basically identical that experimental result shows nano particle, can carry out quantitatively according to a spot of antigen in big or small pair cell surface of fluorescence intensity, thus the early diagnosis of realization disease.Therefore, this fluorescent nano particle can be as a kind of novel biomolecule fluorized marking method, the high sensitivity heterotope that is implemented in biology and medical domain detects, for immeasurable effect is brought into play in single celled original position, on-line analysis, as the detection of unicellular surface antigen, the research of the interior molecular motion track of the tracking of acceptor and cell etc.
Embodiment 2:
(1) preparation of the stock solution of Cd and Se: in the drying box of anhydrous and oxygen-free, 0.2g (0.0025mol) Se is dissolved among the TOP of 4.5ml, the Me of 0.25ml (0.0035mol)
2Cd is added in the TOPSe solution, and with the TOP of 19.5ml dilution, standby.
(2) preparation of the stock solution of Zn and S: same in the drying box of anhydrous and oxygen-free, with (TMS) of 0.52ml (0.0025mol)
2S is dissolved among the TOP of 4.5ml, adds the Me of 3.5ml (0.0035mol)
2Zn solution, with the 16mlTop dilution, standby at last.
(3) the CdSe-ZnS particle is synthetic: the TOPO of 12.5g is heated to 200 ℃ under vacuum condition, dry under this temperature, the nearly 20min of deoxygenation, temperature is elevated to 350 ℃ under 1atmAr gas condition then, behind temperature stabilization, with the 0.7ml (Se of 0.07m mol, 0.1m the Cd of mol) the Cd/Se/Top stock solution is expelled in the reaction bulb, stop heating, the reaction temperature of mixture just has the CdSe nano particle when dropping to nearly 310 ℃ blank occurs, when temperature arrives 300 ℃, the Zn/S/TOP stock solution is added in the solution react; The mol ratio of whole injection reagent is Cd/Se: Zn/s=1: 4, and mixture was 100 ℃ of continuous stirring 1 hour in the reactor.The absolute methanol separation and purification of a nanometer material, deposit is centrifugal, and with absolute methanol rinsing three times, with the unnecessary TOPO of flush away.Nanoparticulate dispersed is in the 10ml anhydrous chloroform, and residue that centrifugal removal is unnecessary and unreacted reagent obtain the stable CdSe nano particle of ZnS thus.
(4) adopt water in oil mode to wrap one deck shell the CdSe nano particle for preparing, its preparation method is: add thiacyclohexane, hexanol, Trtonx-100 and ethyl orthosilicate and ammoniacal liquor in the suspension of CdSe, continuous stirring 24 hours, with acetone separation, centrifugal, with ethanol, water washing, centrifugal, promptly obtain CdSe nano particle with the quartz container parcel.
The present invention also can adopt the correlation technique of prior art to prepare other nano particle of the present invention.
Description of drawings:
Fig. 1 is the transmission electron microscope photo of fluorescent nano particle;
Fig. 2 is the fluorescence spectrum figure of different volumes particle suspension liquid, a wherein, and b, c, d, e are respectively 3ml, 2.5ml, 2ml, 1.5ml, 1ml;
Fig. 3 is fluorescent material Ru (bpy)
36H
2The fluorescence spectrum of O;
Fig. 4 is the fluorescence spectrum of fluorescent nano particle;
Fig. 5 is the fluorescence spectrum of the fluorescent nano particle of sessile antibody;
Fig. 6 is that oxygen content is to fluorometric reagent Ru (bpy)
36H
2The influence that O aqueous solution fluorescence is strong, wherein A is the saturation of the air, B is nitrogen (N
2) saturated;
Fig. 7 is the influence of oxygen content to the fluorescent nano particle fluorescence intensity, and A is the saturation of the air, and B is nitrogen (N
2) saturated;
Fig. 8 and Fig. 9 are the ordinary light source imaging after bone-marrow-derived lymphocyte is cultivated with the immunofluorescence nano particle that is fixed with the goat anti-human igg antibody;
Figure 10 and Figure 11 are the fluorescence imaging after bone-marrow-derived lymphocyte is cultivated with the immunofluorescence nano particle that is fixed with the goat anti-human igg antibody;
Figure 12 is the ordinary light source imaging after bone-marrow-derived lymphocyte is cultivated with the fluorescent nano particle of no antibody;
Figure 13 is the fluorescence imaging after bone-marrow-derived lymphocyte is cultivated with the fluorescent nano particle of no antibody.
Claims (3)
1, a kind of nm-class core-and-shell particles, it is characterized in that, it is a kernel with the nano-substance with one of light, electricity, magnetic characteristic, this kernel is included in and adopts in the package shell that chemistry or physical package package method be prepared from, be combined with by chemical cross-linking agent in the described package shell have chemistry or bio-identification, the active group of combination.
2, nm-class core-and-shell particles according to claim 1, it is characterized in that described kernel is that the inorganic or organic nano of the quantum dot that special fluorescence property is arranged, the magnetic nano particle that the magnetic steering characteristic is arranged, semiconductor nanoparticle, metal nanoparticle, dyestuff micro-capsule and other is built up one of body.
3, nm-class core-and-shell particles according to claim 1 is characterized in that, wraps up medium between described kernel and shell in addition, and this parcel medium adopts the inorganic or organic compound that can evenly wrap up the nano inner core surface and blindly date with shell.
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| CN1183999C true CN1183999C (en) | 2005-01-12 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298807C (en) * | 2002-11-01 | 2007-02-07 | 中国科学院大连化学物理研究所 | Functional nano-rare earth fluorescent micro particle and its preparation and application |
| CN1312479C (en) * | 2003-08-08 | 2007-04-25 | 清华大学 | Nano fluorescent magnetic particle and its preparing method |
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| CN100460338C (en) * | 2007-01-12 | 2009-02-11 | 湖南大学 | Silver/magnetic nanometer particle and its preparation method |
| US20110028662A1 (en) * | 2007-08-31 | 2011-02-03 | Hybrid Silica Technologies, Inc. | Peg-coated core-shell silica nanoparticles and methods of manufacture and use |
| KR100943839B1 (en) * | 2007-10-31 | 2010-02-24 | 한국과학기술연구원 | Method for the production of bio-imaging nanoparticles with high yield by early introduction of irregular structure |
| CN102468026A (en) * | 2010-11-04 | 2012-05-23 | 孙文夫 | Giant magnetic, rectangular magnetic, hard magnetic, hollow and other series bubbles and manufacturing method thereof |
| CN102807225B (en) * | 2011-06-05 | 2015-07-29 | 广州纳科米兹新材料有限公司 | Preparation and the fatty alcohol-polyoxyethylene ether of unordered porous silica silicon materials are applied in this preparation |
| CN102500291B (en) * | 2011-09-30 | 2015-04-08 | 深圳市易瑞生物技术有限公司 | Preparation method and application of magnetic fluorescent nanoparticle with shell-core structure |
| CN102568733B (en) * | 2012-03-02 | 2015-02-25 | 杭州电子科技大学 | Thin-filmed compound broadband anti-electromagnetic interference magnetic powder and preparation method thereof |
| CN104560898B (en) * | 2015-01-26 | 2017-09-22 | 中国农业科学院兰州兽医研究所 | A kind of method that quantum dot is coated with virus-like particle |
| CN104667929B (en) * | 2015-02-10 | 2016-11-30 | 湖南大学 | A kind of magnetic nanometer photocatalyst |
| CN104897889B (en) * | 2015-05-28 | 2017-03-01 | 山东省医学科学院基础医学研究所 | Fluorescence SiO2The preparation method of colloidal agent and use fluorescence SiO2The reagent paper of colloidal agent |
| CN105543395B (en) * | 2016-02-22 | 2019-03-29 | 深圳优圣康医学检验实验室 | Detect the nano-quantum point labelled molecular probes and preparation method thereof of breast cancer HER2 gene |
| CN109929539B (en) * | 2017-12-15 | 2020-07-21 | Tcl科技集团股份有限公司 | Flexible luminescent material and preparation method thereof |
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