Patent Grant
8017036
The present invention relates to a flame synthesis method for synthesizing mono-disperse, highly crystalline phosphorescent oxide nanoparticles with a particle size on the order of less than 10 nm. The present invention also relates to oxide nanoparticles prepared by the inventive flame synthesis method.
Up-converting phosphor (UCP) nanoparticles based on rare-earth doped oxides have recently attracted substantial interest in both academia and industry for the purpose of labeling biological specimens. A UCP is a material which when excited in the infrared, for example with a low intensity CW diode laser, emits a number of narrow emission lines in the visible or near infrared. Because the emitted light is up-converted to shorter wavelengths than the excitation wavelength, background scattering is virtually non-existent. However, achieving mono-disperse particles with a combination of sub-10 nm size, low aggregation, size uniformity and high crystallinity required for high up-conversion efficiency and efficient surface functionalization has proven elusive to the present.
The main advantages provided by UCP nanoparticle imaging are the lack of auto-fluorescence by the specimen, the absence of bleaching, multicolor imaging and the ability to perform high resolution cathodoluminescent imaging with a Scanning Electron Microscope (SEM). Currently a great impediment to using UCP's in biological labeling applications is their size, a point in which quantum dots and organic dyes have excelled. Solution-stable, unaggregated, bright up-converting, oxide-based phosphors have previously been demonstrated for sub-micron sizes.
Because of the high degree of crystallinity required for up-conversion, attempts to synthesize smaller oxide-based up-converting particles were hindered by the need for post-synthesis annealing at a temperature above 500° C., which often leads to undesired sintering of particles. However, the annealing step is not required for the non-up-converting UV or near UV excited phosphors.
Thus, while particles around 10-17 nm based on upconverting NaYF4 have recently been synthesized by sol-gel chemistry approaches, the phosphors are not water-soluble, and the toxicity is as yet undetermined. There remains a need for a method for producing highly crystalline mono-disperse up-converting phosphorescent oxide particles with a particle size on the order of less than 10 nm that are capable of forming stable suspensions or dissolving in a solvent, especially a biocompatible solvent.
The present invention provides essentially mono-disperse sub-10 nm, low aggregating, up-converting nanophosphors that can be suspended in a solvent at very low aggregation levels. The present invention also provides a method by which such nanoparticles can be made.
According to one aspect of the present invention, a method is provided for forming rare earth element doped oxide, oxyhalide and oxysulfide activated nanoparticles by the following method steps:
wherein one or more of the aerosol particle size, flow rate through the Laval tube and pyrolysis temperature are selected to provide a predetermined particle size and degree of crystallinity without particle aggregation.
The present invention also provides methods in which the nanophosphors are imaged inside biological specimens in a 2-photon mode and in a scanning electron microscope (SEM) via cathodoluminescence.
Other features of the present invention will be pointed out in the following description referencing the drawings and claims, which disclose the principles of the invention and the best modes which are presently contemplated for carrying them out.
Sub-10 nm rare earth doped oxide and oxyhalide nanophosphors were synthesized by a flame spray pyrolysis process, using a precursor solution of a host metal salt and dopant salts of rare earth element(s) in a polar solvent. Suitable polar solvents used in the preparation of the precursor solution include, for example, water, ethanol, methanol, isopropanol, n-propanol, n-butanol, hexanol, ethylene glycol, and combinations thereof. The overall combined molar concentration of the oxide- and oxyhalide-forming host metal salt(s) and rare earth element salt(s) in the polar solvent can be from about 0.0001 to about 2.0 M.
The ratio of host metal salts and rare earth element salts in the solution is selected stoichiometrically to provide the desired degree of rare earth element dopant concentration in the nanoparticles. The overall combined concentration is preferably between about 0.01 to about 0.5 M and more preferably between about 0.05 to about 0.1 M. Lower concentration precursor solutions are preferred in order to produce smaller particles.
The water-soluble rare earth element dopants include, but are not limited to, salts represented by the formula:
REX3.yH2O
wherein RE is a rare earth element, y is 4, 5, 6 or 7 and X is an anion forming a water or alcohol soluble salt such as carbonate, hydroxide, halide, nitrate, and the like. Organic compounds capable of forming organometallic complexes cam also be used, such as 2-ethyl-hexanoic acid. Organic complexes are preferred because the compounds provide a fuel source for pyrolysis. Halide salts and other halogen sources are employed stoichiometrically to form oxyhalide salts. Likewise, hydrogen sulfide and other sulfur sources can be used stoichiometrically to form oxysulfide salts. The selection of such source components is readily performed by one of ordinary skill in the art without experimentation when guided by the present specification.
Any rare earth element or combinations thereof can be used (i.e., europium, cerium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, etc.) with europium, cerium, terbium, holmium, erbium, thulium and ytterbium being preferred, and the following combinations also being preferred: ytterbium and erbium, ytterbium and holmium and ytterbium and thulium. Strontium can also be used, and for purposes of the present invention, rare earth elements are defined as including strontium. The oxide, oxyhalide and oxysulfide forming host metal can be, but is not limited to, lanthanum, yttrium, lead, zinc, cadmium, and any of the Group II metals such as, beryllium, magnesium, calcium, strontium, barium, aluminum, radium and any mixtures thereof, or a metalloid selected from silicon and germanium.
The nanophosphors are synthesized using the Laval tube depicted in
The heated Laval tube achieves an ultra-short exposure of the organic gaseous precursor to high temperatures, thereby preventing growth and coalescence of formed nano-particles. The collection end of the Laval tube is fitted with a water-cooled expansion nozzle in order to allow emerging gases to expand and cool rapidly, also to prevent aggregation.
Any reactive gas can be used singularly or in combination to generate the flame for reacting with the precursor solution, such as, but not limited to, hydrogen, methane, ethane, propane, ethylene, acetylene, propylene, butylenes, n-butane, iso-butane, n-butene, iso-butene, n-pentane, iso-pentane, propene, carbon monoxide, other hydrocarbon fuels, hydrogen sulfide, sulfur dioxide, ammonia, and the like, and mixtures thereof. A co-flow of oxygen with the gas may also be employed.
The Laval tube achieves an ultra-short exposure of the synthesized particles to high temperatures and thus suppresses Ostwald ripening and particle aggregation. In the inventive process, the precursor aerosol is ignited in the apex C of the Laval tube, and combustion takes place in the exit cone D. When a methane/oxygen co-flow is used, the temperature of the pyrolysis measured by a type K thermocouple was roughly 1200° C. at a distance of one inch downstream from the apex of the Laval tube. However, any temperature capable of producing the desired degree of crystallinity and particle size without particle aggregation can be employed, which can be readily determined by one of ordinary skill in the art guided by the present specification without undue experimentation.
The desired degree of crystallinity and particle size without particle aggregation can also be attenuated by selection of the appropriate flow rate through the Laval tube and particle size of the atomized droplets entering the tube, the selection of which can also be readily determined by one of ordinary skill in the art guided by the present specification without undue experimentation. One of ordinary skill in the art guided by the present specification will understand how to use one or more of pyrolysis temperature, aerosol particle size and flow rate through the Laval tube to obtain the desired particle size and degree of crystallinity
Rare earth element doped nanoparticles prepared using the Laval tube of the present invention according to the method of the present invention consist essentially of essentially monodisperse particles with a crystallite particle size on the order of less than 10 nm and typically greater than 1 and less than 10 nm. The method of the present invention can be used to prepare the nanoparticles of co-pending and commonly owned U.S. application Ser. Nos. 11/494,157 and 11/537,035 on a size order of less than 10 nm. The disclosures of both applications are incorporated by reference.
Larger nanoparticles can also be prepared by the inventive method. For synthesis of 40 nm particles, rare earth salts are hydrolyzed at a high temperature in a polyol medium in order to reduce hydroxide formation. The resulting solution is heated under a nitrogen atmosphere at 100-200° C., using a heating mantle, with distillation, until the solution appeared clear. The temperature was increased to 40-80° C. and the solution is heated for about 0.2 to about six hours. During this time, the solution changes color as mixed oxides/hydroxides are formed. The glycol based precursor is premixed with a polar solvent such as methanol in order to lower its viscosity prior to spray pyrolysis.
In most biological applications, the nanophosphors have to be surface modified in order to for stable aqueous solutions or dispersions. To that end, the present invention provides silanized poly(alkylene oxide) coatings on the nanophosphors of the present invention, particularly for use with buffer solutions. However, this step is not necessary if the nanophosphors are suspended in non-aqueous solvents.
For silanization, the nanophosphors are dispersed in deionized water by probe tip sonication, followed by 30 ml of methanol and 200 μl of a poly(alkylene oxide)—silane, such as methoxy(polyethyleneoxy)propyltrimethoxysilane (PEG-silane). The suspension is stirred for 0.2 to 5 hours, heated to 40-80° C. for 0.5 to 20 minutes and then cooled. The nano-phosphors are washed, with centrifugation, twice with methanol, twice with methanol:deionized water (1:1) and twice with deionized water, and finally concentrated using centrifugal filter devices. The resulting poly(alkylene oxide)—coated nanophosphors in water were transparent to the eye, and displayed luminescence when excited
The nanoparticles of the present invention enable the production of bio-conjugated near it (NIR) excited luminescent nanoprobes. In particular, enhanced contrast by imaging under NIR excitation where tissue autofluorescence, which occurs under UV excitation, is nonexistent. Nonbleaching and non-blinking UPC probes can be prepared, as compared to fluorescent dye probes that bleach within minutes, especially in high-intensity cell imaging studies.
NIR can penetrate the body up to a depth of several millimeters to centimeters, which accommodates non-invasive probing. Cathodoluminescent emission under electron excitation enables exploitation of electron microscopy techniques in UPC bio-imaging to give enhanced fluorescent imaging resolution through electron microscopy on the order of nano-meters. Hence, bio-labeled UPC's can be employed in multiple mode imaging under UV, NIR and electron excitation. Bio-labeling of UPC's are disclosed in the above-referenced U.S. patent application Ser. Nos. 11/494,157 and 11/537,035. The use of cathodoluminescent imaging improves resolution from roughly 200 nm in conventional optical systems to roughly 5 nm in conventional electron microscopes.
Sub-10 nm sized UPC can be potentially used in subcellular labeling. Both NIR excitable and NIR emitting probes may be developed that enable exciting photons to enter and emitting photons to exit tissue with minimal scattering. The size of the sub-10 nm UPC is on the order of proteins and viruses. Hence, there is the potential of bioconjugating the UPCs for use as sub-cellular bioprobes. The small size should also enable endocytosis.
Nanophosphors were synthesized using a custom-built supersonic flame-spray pyrolysis apparatus. A self-aspirating atomizer (AR27-075-FM02, MicroMist Inc.) was used for formation of a precursor-oxygen aerosol. The premixed precursor was fed into the atomizer at a constant flow rate by a syringe pump (SP101 1, World precision instruments), with an oxygen pressure of 27 psi. The aerosol was ignited by a methane/oxygen flame (CH4=5 psi and O2=4 psi) positioned in front of a alumina laval tube. Nichrome heating wire was coiled around the tube. Oxygen gas (99.99%) and methane gas (˜99%) were purchased from Airgas. The collection end of the laval tube was fitted with a copper water-cooled expansion nozzle in order to allow emerging gases to expand and cool rapidly. Finally, the nanoparticles were collected on a rotating stainless steel cylindrical surface.
For the precursor, 9.6 g of Y(NO3)3.6H2O was dissolved in 50 ml of ethanol and 11.9 ml of 2-ethylhexanoic acid (2-EHA) (99%, Sigma-Aldrich) was added, followed by heating at 60° C. to dissolve. Separately, 11.2 g of Yb(NO3)3.5H2O was dissolved in 50 ml of ethanol with 11.9 ml of 2-ethylhexanoic acid added. The same procedure was performed with 11.1 g of Er(NO3)3.5H2O dissolved in 50 ml of ethanol and 11.9 ml of 2-ethylhexanoic acid. Before use, a 60 ml precursor mixture consisting of 0.1M Yttrium 2-ethylnexanoate (Y(2-EHA)3) solution in ethanol was prepared. To that solution, 0.5 mmoles each of Ytterbium 2-ethylhexanoate (Yb(2-EHA)3) and Erbium 2-ethylhexanoate (Er(2-EHA)3) was added as dopant.
The crystal structure of nanophosphors was determined by x-ray diffraction (XRD) (Rigaku Mini-flex X-ray Diffractometer) over a 2θ range of 15-75 degrees, with a step size of 0.04 degree and a scan speed of 0.8 degree-min−1. The crystal size d was calculated from the full width at half maximum (FWHM) of the (222) peak using Scherrer's equation. The XRD spectrum (
For 2-photon imaging of C. elegans a similar protocol used in the above-referenced U.S. patent application Ser. No. 11/537,035 was followed for inoculation of the worms and transferring onto slides. For cathodoluminescent (CL) imaging, C. elegans were washed with deionized water after inoculation, centrifuged gently to concentrate the worms, pipetted onto a thin layer of dilute Poly-L-lysine (9% by volume) on a gold/silicon substrate, and allowed to dry overnight. Dehydration was performed through a series of ethanol/water mixtures, beginning with 25%, 50% and 100% ethanol (anhydrous, 200 proof, 99.5˜, Sigma Aldrich). About 50 microliters of ethanol/water mixture was applied each time, followed by air drying before the next application. The silicon substrates were mounted onto aluminium stubs with the use of carbon tape. Graphite adhesive was also applied to the edges of the substrates in order to enhance charge dissipation. The mounted substrates were coated with 4 nm thick iridium in order to prevent charging during imaging. The silicon substrate was coated with a 200 nm thick gold film due to the significant CL of Si/SiO2 under electron beam bombardment.
Imaging of the C. elegans by up-conversion luminescence with IR excitation was performed using a inverted microscope with a 60×, 1.4 N.A. oil immersion microscope objective (Nikon, Melville, N.Y.), coupled to an intensified CCD camera (Princeton Instruments, Trenton, N.J.). The worms were imaged in both bright-field and epi-fluorescence geometries. The latter was enabled by a custom-made fluorescence filter set (Chroma technology, Rockingham, Vt.), and a 20-W infrared LED laser array. The illumination intensity was about 10 W/mm2.
Nanoparticle Analysis
In the HRTEM image, facetted spheroidal nanoparticles with well-defined lattice structures are seen. The measured lattice spacing of the selected nanocrystal is 3.16 Angstroms, consistent with the known lattice spacing of the (222) plane of cubic Y2O3. That orientation also appears to be the most frequently observed lattice orientation among the other nanocrystals shown. The corresponding particle size distribution, calculated from over 220 nanoparticles, is shown in
The X-ray Diffraction (XRD) spectrum in
The disparity of the sizes as measured by TEM and XRD can be attributed to the difference in quality of the nanophosphors, which is due to the different sample collection times during flame spray pyrolysis. For TEM analysis, only a small quantity of nanophosphors was collected directly onto TEM grids for about 15 seconds. In contrast, for XRD analysis, a much larger quantity of nanophosphors was required which had to be collected over several hours during spray pyrolysis. Over the long time period, a slight change in spray conditions may have produced variations in size uniformity. This results in a larger size estimated by XRD analysis. How-ever, as shown by TEM data, maintaining size uniformity is possible if spray conditions are strictly controlled. This can be achieved, for example, by installing mass flow controllers to ensure constant flame temperature and aerosol droplet size.
Elemental analysis was performed on the flame spray pyrolysed upconverting nanophosphors by Energy-dispersive X-ray spectrometry (EDX) on a PGT-IMIX PTS EDX system. The sub-10 nm phosphor showed preferential Yb (8.4 atomic percent) doping over Er (6.8 atomic percent) doping.
The viability of the PEG-coated nanophosphors was tested in the digestive system of the nematode worm C. elegans.
More specifically,
Apart from the common problem of sintering in high-temperature synthesized materials, further aggregation is likely to occur when nanoparticles are suspended in solution. The resulting highly aggregated nanophosphors would be rendered impractical for further processing such as bioconjugation. We investigated this issue by suspending as-synthesized nanophosphors in tetrahydrofuran (THF), with and without stabilisation, and measuring the size using dynamical light scattering (
A systematic study was carried out (not shown), whereby the concentrations of the SDS and the upconverting nanophosphors were varied, showed that at about 0.01M SDS in THF, aggregation of the nanophosphors can be reproducibly reduced. We also found that SDS stabilization does not work as well in methanol. Typically, about 1 to 2 mg of nanophosphors were added to the SDS/THF solution, and stirred overnight. The solution was then allowed to equilibrate for several hours before use. When stored over several days sometimes the SDS was observed to settle to the bottom of the dispersion. In that case, samples for sizing measurements were obtained from the clear portion of the dispersion without centrifugation or filtering.
We found that in the absence of SDS, particles agglomerate into clusters of size 100 nm or more. When SDS was used, no further peaks were observed in the 100 nm to 1000 nm range. Typically, at 0.01M SDS and nanophosphor concentration of 0.01 mg/ml, a peak located at roughly 10 nm was obtained, close to that determined using HRTEM and XRD. Although the distribution indicates a small amount of nanoparticles below 10 nm in size, the rest appears to be consistent with two nanoparticle or larger clusters. A control experiment using SDS in THF solution only, confirmed that the UCP/SDS/THF distribution can indeed be attributed to the nanophosphors. These dispersion experiments in THF, demonstrate both a low level of aggregation in our as-synthesized nanophosphors and also facilitate further surface fictionalization work in the near future.
The description of the preferred embodiments should be taken as illustrating, rather than as limiting, the present invention as defined by the claims. As will be readily appreciated, numerous combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the spirit and scope of the invention, and all such modifications are intended to be included within the scope of the following claims.
The present application claims priority benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/894,682 filed Mar. 14, 2007, the disclosure of which is incorporated herein by reference.
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of grant DMR-0213706 awarded by the National Science Foundation.
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