CN102735659A - Nanoparticles used for biological detection with fluoride lutecium as main component - Google Patents
Nanoparticles used for biological detection with fluoride lutecium as main component Download PDFInfo
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Abstract
The invention relates to an application of nanoparticles with fluoride lutecium as a main component in biological detection and preparation of a reagent for biological detection. Furthermore, the invention also relates to the nanoparticles used in the above application and a preparation method thereof. The dimension of the material is less than 200nm. The material has an excellent luminescence property and can be used in bioimaging and biodetection as a luminescent probe.
Description
Technical field
The invention belongs to technical field of biological, particularly, the present invention relates to the lutetium fluoride is the nano particle of principal ingredient is used for the reagent of biological detection in biological detection and preparation application.In addition, the invention still further relates to nano particle of using in the above-mentioned application and preparation method thereof.
Background technology
Rare earth material (also claiming rare earth nano material) with nanoscale transforms the Illuminant nanometer material because of its 4f electronic structure with uniqueness shows and can absorb two or more low-energy photons (near infrared light or infrared light) and launch a high-octane photon (ultraviolet light or visible light) on unique light, electricity, the magnetic property, particularly rare earth.Transforming the Illuminant nanometer material on the rare earth is made up of host material, luminescence rare earth ion and sensitization rare earth ion usually.Wherein, main rare earth ion has Yb
3+, Er
3+, Ho
3+, Sm
3+, Pr
3+And Tm
3+Thereby can form nano level material with a large amount of salt material formation host materials that becomes.The synthetic at present up-conversion luminescence nano material overwhelming majority is the Yb/Er that mixed, Yb/Tm, and Yb/Ho is as the composition of nano material middle rare earth element.
Yet the inventor finds although these materials have obtained application aspect imaging, in biological (imaging) detects, sizable limitation is arranged but.The inventor finds; For biological detection; Particularly for high-sensitive living imaging, existing rare earth nano material water-soluble, have certain limitation with biological cell or the compatibility of molecule and luminous penetration depth etc., thereby when being applied to field of biological detection; Rare earth nano material preferably has following characteristic: size is evenly and less than 200nm; Have good water-solubility, be easy to be connected with biomolecule or absorbed, and have good up-conversion luminescence character by biological (cell).
For this reason, the inventor is through studying for a long period of time, jumped out prior art size is mainly concentrated on the framework to the research of elements such as Y, La, Ca, Sr, Gd less than the rare earth material of 200nm; Found with the lutetium fluoride to be that the nano particle of principal ingredient is used for biological detection surprisingly; It provides new, the better nanoparticle size of luminosity evenly and less than 200nm, has good water-solubility, is easy to be connected with biomolecule or by biological (cell) absorption; Not only realized living imaging; And have good penetration depth, and greatly reduce the background fluorescence of biosome, under identical exciting, can realize many marks.
Summary of the invention
The object of the present invention is to provide with the lutetium fluoride is the technical scheme that the nano particle of principal ingredient is used for biological detection.In addition, the present invention also aims to provide nano particle used in the technique scheme and with cell hatch thing etc. altogether.
Particularly,, the invention provides nano particle that particle diameter is not more than 200nm and in biological detection or in preparation, be used for the application of the reagent of biological detection in first aspect, wherein,
REE in the said nano particle (being X) comprises Lu and Yb, and comprises and be selected among Tm, Er, Dy, Pr and the Ho at least aly, and does not comprise other REEs;
And to account for the molar percentage of REE, Lu accounts for 53-90% (being preferably 55-88%), and Yb accounts for 8-40% (being preferably 10-40%), is selected from REE at least a among Tm, Er, Dy, Pr and the Ho and accounts for 0-8% (being preferably 1-5%).
REE included in the nano particle makes up as stated, is main with Lu.Particularly, said nano particle is to be selected from NaXF
4Nano particle, LiXF
4Nano particle, KXF
4Nano particle, XOF nano particle and XF
3One or more nano particle in the nano particle, wherein X is that above-mentioned REE is formed, and promptly X is a REE, and it comprises Lu and Yb, and comprises and be selected among Tm, Er, Dy, Pr and the Ho at least aly, and does not comprise other REEs.In X, in molar percentage, Lu accounts for 53-90% (being preferably 55-88%), and Yb accounts for 8-40% (being preferably 10-40%), is selected from REE at least a among Tm, Er, Dy, Pr and the Ho and accounts for 0-8% (being preferably 1-5%).
Preferably in first aspect of the present invention, REE in the nano particle and molar percentage thereof are selected from group as shown in the table:
| Group element | Lu | Yb | Tm | Er | Dy | Pr | Ho |
| Group 1 | 79 | 20 | 1 | 0 | 0 | 0 | 0 |
| |
88 | 10 | 0.4 | 1.6 | 0 | 0 | 0 |
| Group 3 | 65 | 30 | 0 | 0 | 5 | 0 | 0 |
| Group 4 | 55 | 40 | 0 | 0 | 0 | 5 | 0 |
| Group 5 | 81 | 15 | 0 | 0 | 0 | 0 | 4 |
| Group 6 | 78 | 20 | 0 | 2 | 0 | 0 | 0 |
In this article, biological detection contains scope understood by one of ordinary skill in the art, comprise organ or tissue's imaging in the cell imaging, animal body (as, liver, spleen or lymph node imaging) or biomacromolecule (as, DNA) detect.In embodiment of the present invention, preferred biological detection is organ or tissue's imaging in cell imaging, the animal body, and wherein animal is a living animal.
Preferably in first aspect of the present invention, what said reagent comprised nano particle, double-layer nanometer particle, water-soluble modified nano particle or their arbitrary and cell hatches thing altogether.The cell of in this article, to hatch thing altogether be cell in the nutrient culture media that contains nano particle, double-layer nanometer particle or water-soluble modified nano particle, cultivated absorption that the back obtains nano particle, double-layer nanometer particle or water-soluble modified nano particle.Preferred said cell is a cancer cell.In addition, also preferred nano particle is double-layer nanometer particle or water-soluble modified nano particle.
Preferably in first aspect of the present invention, said nano particle can be by hydro-thermal method, pyrolysismethod or solvent-thermal method preparation.For example, the process of Hydrothermal Preparation nano particle comprises,
Described rare earth element ion salt, the carboxylic acid that is selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, second alcohol and water are evenly mixed, add LiF, NaF, KF or NH subsequently
4The F WS, the back that stirs are in 130-220 ℃ of hydrothermal treatment consists, and the cooling back adds the organic solvent that is selected from chloroform, ethanol and/or normal hexane, collecting precipitation, and the washing back is dry.
And for example, the process that pyrolysismethod prepares nano particle comprises,
The trifluoroacetate of described rare earth element ion is scattered in the mixed solvent of the carboxylic acid, oleyl amine and the octadecylene composition that are selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid; After moisture content is wherein removed in intensification; In 250 ~ 340 ℃ of cracking trifluoroacetates; The cooling back adds cyclohexane, ultrasonic dispersion and collecting precipitation, and the washing back is dry.
Also as, the process that solvent-thermal method prepares nano particle comprises,
With described rare earth element ion salt, be selected from the carboxylic acid and the octadecylene of oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, add alkali and the NH that is selected from LiOH, NaOH and KOH subsequently
4The F alcoholic solution heats up and to remove alcohol wherein, again under inert gas shielding in 250 ~ 340 ℃ of reactions, the cooling back adds toluene, collecting precipitation, the washing back is dry.
Preferably in first aspect of the present invention, the exemplary preparation method of double-layer nanometer particle comprises,
Get nanoparticulate dispersed in oleic acid and the alkali, water and the alcohol that are selected from LiOH, KOH and NaOH mix, add LuCl again
3The WS stirs, and adds the NaF or the KF WS subsequently again and stirs, and then in 170-230 ℃ of thermal treatment 5-20 hour, the cooling back adds normal hexane, and centrifugal collecting precipitation adds ethanol again and separates out deposition and centrifuging deposition, and the washing precipitation final vacuum is dry.
Preferably in first aspect of the present invention, the preparation method of water-soluble modified nano particle comprises,
Get nanoparticulate dispersed in cyclohexane, add alcohol, water and K
2CO
3, add Lemieux-von Rudloff reagent behind the mixing again and (contain 5.7 mM KMn0
4With 0.105 M NaIO
4The WS), stirred 24-60 hour in 35-45 ℃ then, cooling back centrifugal collecting precipitation, the washing precipitation final vacuum is dry.
In second aspect, the invention provides the application that is used for first aspect present invention nano particle, double-layer nanometer particle or water-soluble modified nano particle or their arbitrary and cell hatch thing altogether.The particle diameter of said nano particle, double-layer nanometer particle or water-soluble modified nano particle is not more than the nanometer of 200nm.
REE in the said nano particle (being X) comprises Lu and Yb, and comprises and be selected among Tm, Er, Dy, Pr and the Ho at least aly, and does not comprise other REEs;
And to account for the molar percentage of REE, Lu accounts for 53-90% (being preferably 55-88%), and Yb accounts for 8-40% (being preferably 10-40%), is selected from REE at least a among Tm, Er, Dy, Pr and the Ho and accounts for 0-8% (being preferably 1-5%).
REE included in the nano particle makes up as stated, is main with Lu.Particularly, said nano particle is to be selected from NaXF
4Nano particle, LiXF
4Nano particle, KXF
4Nano particle, XOF nano particle and XF
3One or more nano particle in the nano particle, wherein X is that above-mentioned REE is formed, and promptly X is a REE, and it comprises Lu and Yb, and comprises and be selected among Tm, Er, Dy, Pr and the Ho at least aly, and does not comprise other REEs.In X, in molar percentage, Lu accounts for 53-90% (being preferably 55-88%), and Yb accounts for 8-40% (being preferably 10-40%), is selected from REE at least a among Tm, Er, Dy, Pr and the Ho and accounts for 0-8% (being preferably 1-5%).
Preferably in second aspect of the present invention, REE in the nano particle and molar percentage thereof are selected from group as shown in the table:
| Group element | Lu | Yb | Tm | Er | Dy | Pr | Ho |
| Group 1 | 79 | 20 | 1 | 0 | 0 | 0 | 0 |
| |
88 | 10 | 0.4 | 1.6 | 0 | 0 | 0 |
| Group 3 | 65 | 30 | 0 | 0 | 5 | 0 | 0 |
| Group 4 | 55 | 40 | 0 | 0 | 0 | 5 | 0 |
| Group 5 | 81 | 15 | 0 | 0 | 0 | 0 | 4 |
| Group 6 | 78 | 20 | 0 | 2 | 0 | 0 | 0 |
Preferably in second aspect of the present invention, said nano particle is by hydro-thermal method, pyrolysismethod or solvent-thermal method preparation.For example, nano particle is by Hydrothermal Preparation, and its preparation process comprises,
Described rare earth element ion salt, the carboxylic acid that is selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, second alcohol and water are evenly mixed, add LiF, NaF, KF or NH subsequently
4The F WS, the back that stirs are in 130-220 ℃ of hydrothermal treatment consists, and the cooling back adds the organic solvent that is selected from chloroform, ethanol and/or normal hexane, collecting precipitation, and the washing back is dry.
And for example, nano particle is by pyrolysismethod preparation, and its preparation process comprises,
The trifluoroacetate of described rare earth element ion is scattered in the mixed solvent of the carboxylic acid, oleyl amine and the octadecylene composition that are selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid; After moisture content is wherein removed in intensification; In 250 ~ 340 ℃ of cracking trifluoroacetates; The cooling back adds cyclohexane, ultrasonic dispersion and collecting precipitation, and the washing back is dry.
Also as, nano particle is by solvent-thermal method preparation, its preparation process comprises,
With described rare earth element ion salt, be selected from the carboxylic acid and the octadecylene of oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, add alkali and the NH that is selected from LiOH, NaOH and KOH subsequently
4The F alcoholic solution heats up and to remove alcohol wherein, again under inert gas shielding in 250 ~ 340 ℃ of reactions, the cooling back adds toluene, collecting precipitation, the washing back is dry.
Preferably in second aspect of the present invention, the double-layer nanometer particle is by being prepared as follows the method preparation, and its preparation process comprises,
Get nanoparticulate dispersed in oleic acid and the alkali, water and the alcohol that are selected from LiOH, KOH and NaOH mix, add LuCl again
3The WS stirs, and adds the NaF or the KF WS subsequently again and stirs, and then in 170-230 ℃ of thermal treatment 5-20 hour, the cooling back adds normal hexane, and centrifugal collecting precipitation adds ethanol again and separates out deposition and centrifuging deposition, and the washing precipitation final vacuum is dry;
Preferably in second aspect of the present invention, water-soluble modified nano particle is by being prepared as follows the method preparation, and its preparation process comprises,
Get nanoparticulate dispersed in cyclohexane, add alcohol, water and K
2CO
3, add Lemieux-von Rudloff reagent behind the mixing again and (contain 5.7 mM KMn0
4With 0.105 M NaIO
4The WS), stirred 24-60 hour in 35-45 ℃ then, cooling back centrifugal collecting precipitation, the washing precipitation final vacuum is dry.
Preferably in second aspect of the present invention, the cell of said to hatch thing altogether be cell in the nutrient culture media that contains nano particle, double-layer nanometer particle or water-soluble modified nano particle, cultivated absorption that the back obtains nano particle, double-layer nanometer particle or water-soluble modified nano particle.Wherein, preferred said cell is a cancer cell.
In the third aspect, the invention provides the preparation method of the said nano particle of second aspect present invention, double-layer nanometer particle or water-soluble modified nano particle.Wherein, the preparation method of said nano particle comprises hydro-thermal method, pyrolysismethod or solvent-thermal method.For example, the Hydrothermal Preparation method of nano particle comprises,
Described rare earth element ion salt, the carboxylic acid that is selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, second alcohol and water are evenly mixed, add LiF, NaF, KF or NH subsequently
4The F WS, the back that stirs are in 130-220 ℃ of hydrothermal treatment consists, and the cooling back adds the organic solvent that is selected from chloroform, ethanol and/or normal hexane, collecting precipitation, and the washing back is dry.
And for example, the pyrolysismethod preparation method of nano particle comprises,
The trifluoroacetate of described rare earth element ion is scattered in the mixed solvent of the carboxylic acid, oleyl amine and the octadecylene composition that are selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid; After moisture content is wherein removed in intensification; In 250 ~ 340 ℃ of cracking trifluoroacetates; The cooling back adds cyclohexane, ultrasonic dispersion and collecting precipitation, and the washing back is dry.
Also as, the solvent-thermal method preparation method of nano particle comprises,
With described rare earth element ion salt, be selected from the carboxylic acid and the octadecylene of oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, add alkali and the NH that is selected from LiOH, NaOH and KOH subsequently
4The F alcoholic solution heats up and to remove alcohol wherein, again under inert gas shielding in 250 ~ 340 ℃ of reactions, the cooling back adds toluene, collecting precipitation, the washing back is dry.
For another example, the preparation method of double-layer nanometer particle comprises,
Get nanoparticulate dispersed in oleic acid and the alkali, water and the alcohol that are selected from LiOH, KOH and NaOH mix, add LuCl again
3The WS stirs, and adds the NaF or the KF WS subsequently again and stirs, and then in 170-230 ℃ of thermal treatment 5-20 hour, the cooling back adds normal hexane, and centrifugal collecting precipitation adds ethanol again and separates out deposition and centrifuging deposition, and the washing precipitation final vacuum is dry.
For example, the preparation method of water-soluble modified nano particle comprises,
Get nanoparticulate dispersed in cyclohexane, add alcohol, water and K
2CO
3, add Lemieux-von Rudloff reagent behind the mixing again and (contain 5.7 mM KMn0
4With 0.105 M NaIO
4The WS), stirred 24-60 hour in 35-45 ℃ then, cooling back centrifugal collecting precipitation, the washing precipitation final vacuum is dry.
The present invention has following beneficial effect:
The nano material uniform particle diameter property that fluoride with lutetium of the present invention is the master is good, and less than 200nm, good biocompatibility is applicable to biological detection.
Nano material of the present invention has good up-conversion luminescence performance than same type of material; Can be good at realizing cell marking; And after advancing the labeled cell hypodermic injection in the nude mouse, detectability is low to moderate about 50 cells, is the highest detection sensitivity of the nano material of prior art.
Palm part through tail vein or mouse is injected in the body, can very clearly observe material and arrive the liver spleen of mouse and the position of lymph, can in living animal, move good biocompatibility.
Hypodermic injection through chest is advanced to deceive in the body of mouse; Observe the upper conversion imaging signal from tow sides respectively; Signal can penetrate black mouse; Other one side at injection point is observed, and the thick about 2.0cm of health of black mouse, this is the highest penetration depth of reporting up to now of this type of nano material signal.
It is narrow that this type of material has emission in addition, and the life-span is long, and advantages such as bleach-resistant have great application value at biomarker and imaging side mask.
The present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is the same excessively in this article for their full text.
For the ease of understanding, below will the present invention be described in detail through concrete embodiment.What need particularly point out is that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this instructions, many variations of the present invention, change all are conspicuous concerning one of ordinary skill in the art.
Description of drawings
Fig. 1: (a) NaXF
4(X=79mol%Lu, 20mol%Yb 1mol%Tm) mix the conversion luminescence spectrum of nano particle under the 980nm laser instrument excites; (b) NaXF
4(X=79mol%Lu, 20mol%Yb, 1mol%Tm) the transmission electron microscope picture of mixing nano particle.
Fig. 2: (a) LiXF
4(X=88mol%Lu, 10mol%Yb, 1.6mol%Er, 0.4 mol%Tm) mixes the conversion luminescence spectrum of nano particle under the 980nm laser instrument excites; (b) LiXF
4(X=88mol%Lu, 10mol%Yb, 1.6mol%Er, 0.4 mol%Tm) mixes the transmission electron microscope picture of nano particle.
Fig. 3: (a) KXF
4(X=65mol%Lu, 30mol%Yb, 5%mol Dy) mixes the transmission electron microscope picture of nano particle; (b) XOF (X=55mol%Lu, 40mol%Yb, 5mol%Pr) the transmission electron microscope picture of mixing nano particle; (c) XF
3(X=81mol%Lu, 15mol%Yb, transmission electron microscope picture 4mol%Ho); (d) parcel NaLuF
4The NaXF of shell
4(X=79mol%Lu, 20mol%Yb, 1mol%Tm) the transmission electron microscope picture of double-layer nanometer particle.
Fig. 4: (a) NaXF
4(X=78mol%Lu, 20mol%Yb 2mol%Er) mix the conversion luminescence spectrum of nano particle under the 980nm laser instrument excites; (b) NaXF
4(X=78mol%Lu, 20mol%Yb, 2mol%Er) the transmission electron microscope picture of mixing nano particle.
Fig. 5: the cell imaging photo, wherein scale is 30 μ m.
Fig. 6: (a) cell skin of absorption nano particle is made a bet and is injected the imaging picture in the mouse body; (b) the nano particle tail vein injection advances and is scattered in the image pattern sheet in the mouse body; (c) the nano particle forefoot is injected into the lymph imaging picture in the mouse body.
Fig. 7: the picture of nano particle penetration depth in black mouse body, (a) be mouse chest face, (b) be the mouse back side.
Embodiment
Raw material used in the present embodiment is known compound, can be obtained by commercial sources, maybe can prepare by means known in the art.In following embodiment, related physical and chemical parameter is by following Instrument measuring:
The size of utilizing JEOL JEM-2010F high resolution transmission electron microscope to observe nano particle.Fluorescence emission spectrum records on the LFS920 of Edinburgh company XRF; Excitation source is the stable state 980nm laser instrument (Beijing Hai Te photoelectricity company) that 0-1W adjustable power, continuous wave excite, and detector is the photomultiplier (PMT) of semiconductor refrigerating.Co-focusing imaging is accomplished on one Chinese patent application 200810038857.7 described conversion illuminating microscope on mesomeric state laser pump.The toy living imaging is accomplished in the described mesomeric state laser pump up-conversion luminescence of Chinese patent CN201394004Y toy living imaging system.
Embodiment 1:NaXF
4
(X=79mol%Lu, 20mol%Yb, 1mol%Tm) preparation of mixing nano particle
Mixed in proportion salt (CF
3COO)
3(1mol%Tm) totally 2 mmol add the sodium trifluoroacetate of 4 mmol to X for X=79mol%Lu, 20mol%Yb; Be scattered in the 20ml oleyl amine, be warming up to 110 ℃ of 1h that dewater, be warming up to 340 ℃ then, constant temperature 2h; Be cooled to room temperature then, add 30 ml cyclohexanes, disperse 5min with 80% power with KQ-500DB type ultrasonic generator; With the centrifugal 10min of 14000r/min, discard the liquid part then, precipitated solid is with 3 times (each 10 ml) of cyclohexane washing.Solid after washing vacuum drying at room temperature 12 hours promptly obtains NaXF
4(X=79mol%Lu, 20mol%Yb 1mol%Tm) mix nano particle.Prepare practical nano level particle through identifying that shown in Fig. 1 b, the particle diameter of this nano particle is about 20nm, and the basic homogeneous of particle diameter, showing.
Identify this nano particle with 980nm laser as excitaton source; The result finds that this nano particle demonstrates typical ultraviolet light (350-375nm), blue light (460-495nm), ruddiness (630-660nm and 680-710nm) and infrared light (770-820nm) shown in Fig. 1 a.
Embodiment 2:LiXF
4
(X=88mol%Lu, 10mol%Yb, 1.6mol%Er, 0.4 mol%Tm) mixes the preparation of nano particle
Mixed in proportion rare-earth chlorination salt XCl
3(X=88mol%Lu, 10mol%Yb, 1.6mol%Er; 0.4 mol%Tm) be total to 1mmol, be scattered in the middle of the mixed solution of 10mL oleic acid and 10 mL octadecylenes, be heated to 160 ℃ of solution that form homogeneous; Cool to room temperature adds the NH that 10mL contains 2 mmol LiOH and 4 mmol
4The methanol solution of F stirs 30min, is warming up to 100 ℃ and removes methyl alcohol 1h, under the Ar gas shield, is warmed up to 300 ℃ of reactions 1 hour again; Cool to room temperature adds 25 ml toluene, with the centrifugal 8min of 12000rpm; Discard the liquid part, disperse washing also centrifugal 3 times with toluene and (suspend with 10 ml toluene at every turn and precipitate, then with the centrifugal 5min of 12000rpm; Discard the liquid part), precipitated solid vacuum drying at room temperature 24 hours promptly obtains LiXF
4(X=88mol%Lu, 10mol%Yb, 1.6mol%Er, 0.4 mol%Tm) mixes nano particle.Prepare practical nano level particle through identifying that shown in Fig. 2 b, the particle diameter of this nano particle is about 40nm, and the basic homogeneous of particle diameter, showing.
Identify this nano particle with 980nm laser as excitaton source, the result shown in Fig. 2 a, find this nano particle appear typical green glow (520 ~ 570nm), ruddiness (650 ~ 670nm) with near infrared (switching emission on 755 ~ 840nm).
Embodiment 3:KXF
4
(X=65mol%Lu, 30mol%Yb, 5%mol Dy) mixes the preparation of nano particle
1.2g KOH, 9ml water, 10ml ethanol and 20ml oleic acid are mixed, stir and obtain uniform solution, in this solution, add the mixed in proportion rare earth-iron-boron XCl that 1.2ml amounts to 0.5mol/L
3(WS 5mol%Dy) stirs 5min, adds 4ml NaF (1.0M) WS subsequently again, stirs 10min for X=65mol%Lu, 30mol%Yb.Mixed solution is transferred in the 50ml water heating kettle, in 160 ℃ of hydrothermal treatment consists 16h.Add 20 ml normal hexanes behind the natural cooling, dissolution precipitation adds 20 ml ethanol and separates out deposition and centrifuging collection, and deposition is deposited in-30 ℃ of following vacuum drying 10 hours with 3 times (each 10 ml) of normal hexane washing, promptly obtains KXF
4(X=65mol%Lu, 30mol%Yb, 5%mol Dy) mixes nano particle.Prepare practical nano level particle through identifying that shown in Fig. 3 a, the particle diameter of this nano particle is about 70nm, and the basic homogeneous of particle diameter, showing.
Embodiment 4:XOF (X=55mol%Lu, 40mol%Yb, 5mol%Pr) preparation of mixing nano particle
Mixed in proportion (CF
3COO)
3(5mol%Pr) 1mmol is scattered in the middle of the 10ml oleyl amine X for X=55mol%Lu, 40mol%Yb; Be warming up to 90 ℃ of 1.5h that dewater, be warming up to 330 ℃ then, constant temperature 3h; Be cooled to room temperature, add 25 ml normal heptanes, disperse 10 min with 80% power with KQ-500DB type ultrasonic generator; With the centrifugal 8min of 10000r/min, discard the liquid part, precipitated solid is with 3 times (each 10 ml) of normal heptane washing.Be deposited in 50 ℃ of following vacuum drying 2 hours after the washing, (X=55mol%Lu, 40mol%Yb 5mol%Pr) mix nano particle promptly to obtain XOF.Prepare practical nano level particle through identifying that shown in Fig. 3 b, the particle diameter of this nano particle is about 3nm, and the basic homogeneous of particle diameter, showing.
Embodiment 5:XF
3
(X=81mol%Lu, 15mol%Yb, 4mol%Ho) preparation of mixing nano particle
9ml water, 10ml ethanol and 20ml oleic acid are mixed, stir and obtain uniform solution, in this solution, add the mixed in proportion rare earth-iron-boron XCl of the 0.5mol/L that amounts to 1.2ml
3(WS 4mol%Ho) stirs 5min, adds 4ml 1.0M NH subsequently again for X=81mol%Lu, 15mol%Yb
4The F WS stirs 10min.Mixed solution is transferred to the 50ml water heating kettle, in 200 ℃ of hydrothermal treatment consists 48h.Add 20 ml chloroforms behind the natural cooling, dissolution precipitation adds 20 ml acetone and separates out deposition and centrifuging, and deposition is deposited in 10 ℃ of following vacuum drying 48 hours subsequently with 8 ml chloroforms washing 5 times, promptly obtains XF
3(X=81mol%Lu, 15mol%Yb 4mol%Ho) mix nano particle.Prepare practical nano level particle through identifying that shown in Fig. 3 c, the particle diameter of this nano particle is about 200nm, and the basic homogeneous of particle diameter, showing..
Embodiment 6:NaXF
4
(X=78mol%Lu, 20mol%Yb, 2mol%Er) preparation of mixing nano particle
0.6g NaOH, 9ml water, 10ml ethanol, 10ml oleic acid and 4.0g six aminocaproic acids are mixed, stir and obtain uniform solution, in this solution, add the mixed in proportion rare earth-iron-boron XCl that amounts to 0.6ml 1mol/L
3(WS 2mol%Er) stirs 5min, adds the 4ml 1.0M NaF WS subsequently again, stirs 10min for X=78mol%Lu, 20mol%Yb.Mixed solution is transferred to the 50ml water heating kettle, in 200 ℃ of hydrothermal treatment consists 12h.Add 15 ml cyclohexanes behind the natural cooling, dissolution precipitation adds 20 ml ethanol and separates out deposition and centrifuging, and deposition 15 ℃ of following vacuum drying 36 hours, promptly obtains NaXF with 3 times (each 12 ml) of cyclohexane washing
4(X=78mol%Lu, 20mol%Yb 2mol%Er) mix nano particle.Prepare practical nano level particle through identifying that shown in Fig. 4 b, the particle diameter of this nano particle is about 20nm, and the basic homogeneous of particle diameter, showing.
Identify this nano particle with 980nm laser as excitaton source, the result finds that this nano particle presents typical green glow (520 ~ 570nm) and the ruddiness (switching emission on 650 ~ 670nm) shown in Fig. 4 a.
Embodiment 7: parcel NaLuF
4
The NaXF of shell
4
(X=79mol%Lu, 20mol%Yb, 1mol%Tm) preparation of double-layer nanometer particle
The NaXF that embodiment 1 is synthetic
4(X=79mol%Lu, 20mol%Yb 1mol%Tm) mix nano particle 50mg and are distributed in the 20ml oleic acid, mix with 1.2g KOH, 9ml water, 10ml ethanol then, stir and obtain uniform solution, in this solution, add the LuCl of 1.2ml 0.5mol/L
3The WS stirs 5min, adds the 4ml 1.0M NaF WS subsequently again, stirs 10min.Mixed solution is transferred to the 50ml water heating kettle, in 200 ℃ of hydrothermal treatment consists 10h.Add 24 ml normal hexanes behind the natural cooling, dissolution precipitation adds 20 ml ethanol and separates out deposition and centrifuging, with 3 times (each 6 ml) of normal hexane washing, 30 ℃ of following vacuum drying 10 hours, promptly obtains outer parcel NaLuF
4Nucleocapsid, kernel are NaXF
4(X=79mol%Lu, 20mol%Yb, double-layer nanometer particle 1mol%Tm).Through identifying, shown in Fig. 3 d, the particle diameter of this nano particle is about 30nm, than the kernel of embodiment 1 preparation big about 10nm, and the basic homogeneous of particle diameter shows and prepares practical nano level particle.
Embodiment 8: the preparation of the nano particle that the surface is water-soluble modified
The NaXF that the embodiment of 0.5 g of new system 1 is synthetic
4(X=79mol%Lu, 20mol%Yb 1mol%Tm) mix nanoparticulate dispersed in the cyclohexane of 350 ml, add the 180 ml tert-butyl alcohols, the K of 60 ml water and 0.5 g
2CO
3, stir 20 min under the room temperature, and then add 20 ml Lemieux-von Rudloff reagent and (contain 5.7 mM KMn0
4With 0.105 M NaIO
4The WS).Above mixed solution stirs 48 h at 40 ℃.After being cooled to room temperature, separate, discard liquid, then wash twice with ethanol again for 2 times with deionized water wash with centrifugal 10 min of 12,000 rpm.The vacuum drying deposition is spent the night, and obtains the water-soluble modified nano material in surface.Identify this nano particle with 980nm laser as excitaton source, find that this nano particle demonstrates typical ultraviolet light (350-375nm), blue light (460-495nm), ruddiness (630-660nm and 680-710nm) and infrared light (770-820nm).
Embodiment 9: nano particle of the present invention is used for cell imaging
RPMI.1640 nutrient culture media (1X) with the surperficial water-soluble modified nano material that contains 200 μ g/mL embodiment, 7 preparations is hatched KB (human oral cavity epithelial cancer cell; Can be available from the U.S. representative microbial preservation center (ATCC)) cell 2h; Use PBS buffer solution cells washed 3 times then; The material flush away that will do not absorbed by cell, the KB cell after will hatching then forms images on conversion illuminating microscope on mesomeric state laser pump, uses 60 * oily sem observation.The stable state 980nm laser instrument (Beijing Hai Te photoelectricity company) that use 0-1W adjustable power, continuous wave excite in the imaging process is observed the emission light of 470 ± 20nm as exciting light.The result is as shown in Figure 5, and can observe cell can launch macroscopic light under the sightless exciting light of naked eyes, shows that the nano material of being taken in by cell can be used in cell imaging.
Embodiment 10: the imaging in animal body of the cell of nanoparticle label of the present invention
Get 50 in the KB cell that embodiment 9 has absorbed nano material; Advance through hypodermic injection in the body of nude mice; This mouse is placed on carries out the up-conversion luminescence imaging on the conversion living imaging instrument; As exciting light, observations can be observed the signal that stimulated luminescence is clearly arranged in the injection site shown in Fig. 6 a with the laser of 980nm.
Embodiment 11: the imaging of nano particle of the present invention in the mouse body
The surperficial water-soluble modified nano material of getting embodiment 7 preparations is dissolved in the physiological saline; Be mixed with the solution of 2mg/mL; Advance 20 μ L in the Kunming mouse body through tail vein injection, behind the 1h this mouse be placed on and carry out the up-conversion luminescence imaging on the conversion living imaging instrument, with the laser of 980nm as exciting light; The result can observe clearly stimulated luminescence signal in mouse liver and spleen shown in Fig. 6 b.
Embodiment 12: the imaging of nano particle of the present invention in mouse lymph
The surperficial water-soluble modified nano material of getting embodiment 7 preparations is suspended in the physiological saline; Be mixed with the solution of 1mg/mL, the forefoot through mouse is injected into 5 μ L in the mouse body, behind the 10min; This mouse is placed on carries out the up-conversion luminescence imaging on the conversion living imaging instrument; As exciting light, the result can observe clearly stimulated luminescence signal in the axillary lymph of mouse shown in Fig. 6 c with the laser of 980nm.
Embodiment 13: the research of nano particle of the present invention penetration depth in the mouse body
The surperficial water-soluble modified nano material of getting embodiment 7 preparations is suspended in the physiological saline; Be mixed with the solution of 2mg/mL; Advance to deceive 10 μ L in the mouse body through the chest hypodermic injection, this mouse be placed on carry out the up-conversion luminescence imaging on the conversion living imaging instrument, with the laser of 980nm as exciting light; The result is as shown in Figure 7; Can be respectively observe the signal of stimulated luminescence clearly at chest and back, the chest of black mouse is about 2.0cm to the distance at back, so nano particle of the present invention can have very dark penetration depth and can not lose imaging capability in vivo.
Claims (10)
1. the particle diameter nano particle that is not more than 200nm is used for the application of the reagent of biological detection in biological detection or in preparation, wherein,
REE in the said nano particle (being X) comprises Lu and Yb, and comprises and be selected among Tm, Er, Dy, Pr and the Ho at least aly, and does not comprise other REEs;
And to account for the molar percentage of REE, Lu accounts for 53-90%, and Yb accounts for 8-40%, is selected from REE at least a among Tm, Er, Dy, Pr and the Ho and accounts for 0-8%.
2. the described application of claim 1, wherein said nano particle is to be selected from NaXF
4Nano particle, LiXF
4Nano particle, KXF
4Nano particle, XOF nano particle and XF
3One or more nano particle in the nano particle, X are the described REE of claim 1.
3. claim 1 or 2 described application, wherein REE in the nano particle and molar percentage thereof are selected from group as shown in the table:
。
4. the arbitrary described application of claim 1-3, wherein said biological detection comprise organ or tissue's imaging in the cell imaging, animal body (as, liver, spleen or lymph node imaging) or biomacromolecule (as, DNA) detect.
5. the arbitrary described application of claim 1-4; What wherein said reagent comprised nano particle, double-layer nanometer particle, water-soluble modified nano particle or their arbitrary and cell hatches thing altogether; Wherein, said nano particle can be by hydro-thermal method, pyrolysismethod or solvent-thermal method preparation;
Said double-layer nanometer particle can be prepared by following method:
Get nanoparticulate dispersed in oleic acid and the alkali, water and the alcohol that are selected from LiOH, KOH and NaOH mix, add LuCl again
3The WS stirs, and adds the NaF or the KF WS subsequently again and stirs, and then in 170-230 ℃ of thermal treatment 5-20 hour, the cooling back adds normal hexane, and centrifugal collecting precipitation adds ethanol again and separates out deposition and centrifuging deposition, and the washing precipitation final vacuum is dry;
Said water-soluble modified nano particle can be prepared by following method:
Get nanoparticulate dispersed in cyclohexane, add alcohol, water and K
2CO
3, add Lemieux-von Rudloff reagent behind the mixing again and (contain 5.7 mM KMn0
4With 0.105 M NaIO
4The WS), stirred 24-60 hour in 35-45 ℃ then, cooling back centrifugal collecting precipitation, the washing precipitation final vacuum is dry.
6. be used for claim 1-5 arbitrary described nano particle, double-layer nanometer particle or water-soluble modified nano particle or their arbitrary and cell hatch thing altogether.
7. the preparation method of the described nano particle of claim 6, double-layer nanometer particle or water-soluble modified nano particle.
8. the preparation method of the described nano particle of claim 7, it is a hydro-thermal method, comprises,
The described rare earth element ion salt of claim 1, the carboxylic acid that is selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, second alcohol and water are evenly mixed, add LiF, NaF, KF or NH subsequently
4The F WS, the back that stirs are in 130-220 ℃ of hydrothermal treatment consists, and the cooling back adds the organic solvent that is selected from chloroform, ethanol and/or normal hexane, collecting precipitation, and the washing back is dry.
9. the preparation method of the described nano particle of claim 7, it is a pyrolysismethod, comprises,
The trifluoroacetate of the described rare earth element ion of claim 1 is scattered in the mixed solvent of the carboxylic acid, oleyl amine and the octadecylene composition that are selected from oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid; After moisture content is wherein removed in intensification; In 250 ~ 340 ℃ of cracking trifluoroacetates; The cooling back adds cyclohexane, ultrasonic dispersion and collecting precipitation, and the washing back is dry.
10. the preparation method of the described nano particle of claim 7, it is a solvent-thermal method, comprises,
With the described rare earth element ion salt of claim 1, be selected from the carboxylic acid and the octadecylene of oleic acid, linoleic acid, six aminocaproic acids and cinnamic acid, add alkali and the NH that is selected from LiOH, NaOH and KOH subsequently
4The F alcoholic solution heats up and to remove alcohol wherein, again under inert gas shielding in 250 ~ 340 ℃ of reactions, the cooling back adds toluene, collecting precipitation, the washing back is dry.
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