Method of production of fluorescent labels based on biodegradable nanoparticles of silicon for use in vivo

FIELD: nanotechnology.

SUBSTANCE: invention relates to the field of nanomaterials. A method is proposed for production of fluorescent labels based on biocompatible and biodegradable nanoparticles of silicon for application in vivo by the reaction of disproportionation of silicon monoxide at a temperature of 950°C in air atmosphere followed by interaction of the resulting nanoparticles of silicon with dimethylsulfoxide. The proposed method enables to produce two outfits of nanoparticles with the size of 2.0-2.5 nm with photoluminescence peaks at 676 nm and 774 nm.

EFFECT: production of hydrophilic biocompatible and biodegradable fluorescent labels of nanocrystalline silicon, having sustained bright luminescence and narrow function of size distribution, resistant to high temperatures (up to 220°C) without the use of toxic substances in the process of their synthesis; resulting nanoparticles are applicable as labels in vivo in imaging of deep-seated tissues and organs.

1 cl, 3 dwg

 

The invention relates to the field of nanotechnology and nanomaterials, the method of obtaining fluorescent labels based on biocompatible and biodegradable silicon nanoparticles for in vivo applications, allowing you to receive a mass quantity of nanomaterial.

Currently one of the most common approaches to solving this problem is the use of semiconductor quantum dots based on CdSe, CdTe. The wavelength of maximum fluorescence depends on the kernel size and nature of the semiconductor. This requirement satisfies the following semiconductors: CdSe, CdTe, PbSe, PbS, InP. Several yielding the best fluorescent labels in the quantum fluorescence yield (~70% at room temperature), nanocrystals exceed by several orders of magnitude in the values of the absorption cross-section of the exciting light. As a result, the brightness of the nanocrystals is so high that can detect single objects using a standard fluorescent microscope.

Recently developed methods of synthesis photostabilizer nanocrystals InP, InP/ZnS, but the low brightness of the fluorescence and a wide distribution of particle sizes limit their use in biochemistry. The disadvantages of CdSe, CdTe, PbSe, PbS, InP, InP/ZnS toxicity is also included in the composition of the quantum dots ions that will require building a special protective shells. Suspicious attitude toward the use of quantum dots in vivo was recently expressed in a number of works: by ICP MS was prodemonstrirovano accumulation Kanovich points in the body of the animal after administration of intravenous. In addition, nanometer size crystals can lead to passive or active transport and accumulation in cell organelles, leading to unpredictable delayed effects. Therefore, the search for quantum dots with alternative chemical composition and do not contain toxic ions, is extremely important. In this regard, of particular interest are quantum dots based nanogramme.

The closest to the essential features of the claimed method of obtaining fluorescent labels based on biodegradable silicon nanoparticles for in vivo applications is the method proposed in [Park, J.-H., Gu L., von Maltzahn g, Ruoslahti E., S.N. Bhatia, and M.J. Sailor Biodegradable luminescent porous silicon nanoparticles for in vivo applications // Nature materials. 2009. V.8, №4. P.331-336]. In this paper, fluorescent nanoparticles of porous silicon was obtained by the method of electrochemical etching of a silicon substrate in an alcohol solution of hydrofluoric acid, the removal of the porous silicon film, followed by sonication and the allocation obtained porous silicon nanoparticles by filtration through a membrane with the size of the om 0.22 μm pores. To activate fluorescence obtained porous nanoparticles were incubated in water for two weeks. Immediately after forming the porous layer, dangling bonds of silicon on the surface is then passivated mainly hydrogen, which is replaced by oxygen. During the stage of activation, treated with hydrogen since the capacity of silicon oxide. Such structures exhibit strong luminescence associated with defects localized at the interface of silicon and silicon dioxide.

The disadvantages of the invention are: a large particle size is 126 nm and modal distribution of the spectral intensity of fluorescence in the field of 650-900 nm, which does not allow spectral encoding fluorescent labels with different wavelengths of fluorescence in different proportions.

The technical result of the present invention is a method of obtaining a hydrophilic biocompatible and biodegradable fluorescent labels nanocrystalline silicon with a stable bright luminescence with an intensity peaks in the area of 676 nm and 774 nm, resistant to high temperatures (up to 220°C) without the use of toxic substances in the process of their synthesis.

This technical result is achieved due to the fact that to obtain hydrophilic fluorescent the data labels use the disproportionation of silicon monoxide at a temperature of 950°C in air, followed by the interaction of nanoparticles of silicon with dimethylsulfoxide, leading to the formation of two ensembles of nanoparticles with average sizes of 2.0-2.5 nm and a narrow distribution in size from 1.3 to 4.0 nm, having a photoluminescence maxima at 650 nm and 730 nm.

Use the following disproportionation fine silicon monoxide at a temperature of 950°C. to obtain nanocrystalline silicon:

where n is the number of silicon atoms in the silicon nanoparticles (n=29 for silicon nanoparticles of diameter of 1.0 nm; n=286 for silicon nanoparticles of a diameter of 2.0 nm), followed by dissolution and leaching of silica in the acidified aqueous solution.

The formation of nanocrystalline silicon occurs in the thickness of the crystals formed during the sintering of oxide of silicon, similar to the decomposition of solid solutions that effectively protects nanocrystalline silicon from oxidation by air. However, annealing in air leads to the formation of impurity centers (Si=O) and (Si-O-Si), energy levels which lie within the bandgap of the silicon nanoparticles of a size of 2.0-2.5 nm, which allows to shift the maximum of fluorescence required for the fluorescent labels red region of the spectrum. Immediately after annealing see the luminescence of the obtained nanogramme when exposed to UV radiation in the red region of the spectrum.

For stabilizat and and hydrophilization the surface of the silicon nanocrystals annealed mixture in a Teflon beaker pour the concentrated hydrofluoric acid, warmed up slightly and placed in an ultrasonic bath for intensification etching. After dissolution products at 313K solid particles precipitated by centrifugation and washed 2 times with ethanol. Received luminescense sediment Usacheva and transferred into a quartz tube containing dimethyl sulfoxide. The tube is heated to 150°C for complete evaporation of ethanol, again Usacheva in an ultrasonic bath and quickly heated to the boiling point of dimethylsulfoxide (~189°C). During 30 to see the darkening mist and a significant increase in the brightness of a luminescence. The process can be described by the following reaction equation:

where m is the number of hydrogen atoms on the surface of nanoparticles (m<n; m=24 for silicon nanoparticles of diameter 1.0 nm). The basis of Zola are hydrophilic silicon nanoparticles in dimethyl sulfoxide, which is known to be non-toxic substance. Obtained by coagulation of a colloidal solution of silicic nanopure depending on the modes of reaction may consist of particles with a size of up to several nanometers in diameter and has a definite crystalline structure of the Central core (Figure 1 and 2).

Silicon nanoparticles with a size of 2.0-2.5 nm have own intensive luminescence in the orange-red region of the spectrum with maximum and 676 nm and 774 nm (Figure 3). Quantum yield of fluorescence obtained biometar nanoparticles is more than 15%.

From Figure 3 it is evident that the proposed method of producing a fluorescent biometar gives two ensembles of particles danocrine having a fluorescence maximum at 676 nm (curve 2) and 774 nm (curve 3). Fluorescent labels having the luminescence spectrum at different wavelengths allow you to implement a spectral encoded microparticles, which opens the way for the development of test-systems for rapid multi-parameter analysis of a large number of biological objects based on microarray technique. One of the advantages of use as fluorophores NC with different wavelengths issue is the possibility of excitation of all coding components with a single monochromatic source. To obtain a solution of fluorescent labels sample of the original solution is dried under reduced pressure and diluted with distilled water.

The development of a fairly simple method of obtaining a biocompatible and biodegradable fluorescent labels based on nanocrystalline silicon, with a bright stable photoluminescence in the visible spectral range in mass quantities opens the possibility of their application in medicine and biology for fluorescent diagnostics, photodynamic who photothermal therapy photochemical sterilization of the blood supply, as well as in ecology for water purification from organic pollutants and pathological microflora.

The unique optical properties of danocrine, such as photostability and a wide range of bands of fluorescence depending on the diameter of the core nanoparticles, including in the near-infrared range, make them attractive for use as in vivo markers for imaging superficial tissues and organs. Another option is to use the obtained fluorescent labels - flow cytofluorimetry, allowing to analyze the spectral properties of each passing through the detector nanoparticles. The use of spectral encoded nanoparticles involves the analysis of each fluorescent label for detection of the presence in the analyzed sample of each of the detected objects.

This invention will find wide application in medicine for the diagnosis of tumors of various types. For example, for imaging of deep tumors in vivo wavelength excitation fluorescence nanoparticles of silicon must be selected in the near-red region of the spectrum in order to obtain maximum absorption of radiation cloth and minimum absorption chromophore such proteins, such as hemoglobin.

The method according to the teachings of fluorescent labels based on biocompatible and biodegradable silicon nanoparticles for in vivo applications, characterized in that for obtaining hydrophilic fluorescent labels use the disproportionation of silicon monoxide at a temperature of 950°C in air, followed by the interaction of nanoparticles of silicon with dimethylsulfoxide, leading to the formation of two ensembles of nanoparticles with a size of 2.0-2.5 nm, with the maximum photoluminescence at 676 nm and 774 nm.



 

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