Patent Application
20090170094
The present invention relates to a fluorescent labeling substance which comprises core/shell nanoparticles or core/shell nanorods and is used for analysis targeting biosubstances.
Nanoparticles are applied to the bio-field as a fluorescent labeling substance used for analysis of the behavior of different genes proteins in a cell which have been dyed with plural colors (as described in non-patent document 1).
When using a semiconductor nanoparticle exhibiting such behavior, the lectin is generally introduced onto the overall particle surface (
There was proposed a semiconductor nanoparticle surface-modified with a compound containing a hydrophilic functional group, for use as a fluorescent labeling substance having enhanced hydrophilicity and causing no coagulation in an aqueous solution. However, such a semiconductor nanoparticle is modified on its entire surface and problems arise with the light-emitting efficiency, as described above.
It is an object of the invention to provide a labeling substance which is capable of realizing more suitable labeling through enhancement of light-emitting efficiency of a semiconductor nanoparticle or nanorod.
The present invention has come into being, based on the discoveries by the inventors of this application that in at least a part of the shell surface of a semiconductor nanoparticle or nanorod having a modification group capable of being adsorbed to a biosubstance, for example, an antigen such as a sugar chain existing on the cancer cell surface, a protein or a nucleic acid, a region of such a modification group being not present was provided, and thereby was obtained a fluorescent labeling substance exhibiting enhanced efficiencies of exciting light entering to and fluorescence emitting from semiconductor nanoparticles or nanorods.
The area of the above-described region having no modification group preferably accounts for not less than 50% of the total shell surface. For example, a modification group capable of adsorbing a biosubstance is allowed to exist on only one half of the spherical surface of a nanoparticle, only one half of the cylindrical surface of a nanorod, or only the upper surface or only the bottom surface of a nanorod, rendering it feasible to secure an area of such a region.
The modification group capable of being adsorbed to a biosubstance is introduced as follows; a COOH (carboxyl) group or a NH2 (amino) group is formed on the surface of a nanoparticle or nanorod by carboxylation (COOH formation) of an alkyl group such as a CH3 group or C having an unbonded bond, contained in SiC, SiOCH, SiCNH or the like or by forming a substance containing a NH2 group, and then, the thus formed carboxy group or amino group is allowed to react with a modification group.
Preferably, the nanoparticle or nanorod of the invention is a core/shell one comprising a core composed of a semiconductor nanocrystal and a shell composed of a substance having a greater band gap than the core, and the average particle diameter of nanoparticles or the average diameter of nanorods is desirably from 2 to 50 nm.
According to the invention, there is provided a fluorescent labeling substance with enhanced substantial light-emitting efficiency and capable of performing highly precise analysis in the field of targeting biosubstances, such as detection of cancer cells.
The present invention relates to a fluorescent labeling substance which comprises core/shell nanoparticles or core/shell nanorods and is used for analysis targeting biosubstances.
Nanoparticles are applied to the bio-field as a fluorescent labeling substance used for analysis of the behavior of different genes proteins in a cell which have been dyed with plural colors (as described in non-patent document 1).
Such an average particle size or average rod diameter can be determined through observation by TEM (transmission electron microscope) and the average of measurement values obtained by observation of at least 200 particle images is employed therefor.
Materials constituting the foregoing nanosized particulates are not specifically limited and examples thereof include I-VII group compound semiconductors such as InAs, II-VI group compound semiconductors such as CdS and CdSe, III-V group compound semiconductor such as InAs, IV group semiconductors such as Si and there can also be optimally chosen crystals of these compound semiconductors. Of these, in the invention, the use of a semiconductor nanoparticle formed of Si is suitable without using materials having concerns regarding environmental pollution or toxicity to the human body and in terms of achieving superior emission. With regard to the core/shell constitution, there can be chosen a suitable combination according to employed semiconductor nanoparticle, for example, CdSe-core/ZnS-shell and Si-shell/SiO2-shell.
In the semiconductor labeling substance of the invention, a modification group to bind specifically to a biosubstance such as a protein, nucleic acid or antigen exists on the surface of the nanosized particulate (the shell surface of a nanoparticle or nanorod). The modification group contains at least a site capable of direct-binding specifically to a biosubstance such as a protein, nucleic acid or antigen (hereinafter, also denoted as biosubstance binding site) and a site directly bound to the surface of the nanosized particulate (hereinafter, also denoted as surface binding site), which may further contain an intermediate site linking the biosubstance binding site and the surface binding site. The fluorescent labeling substance of the invention becomes capable of be bound to a biosubstance as a target of labeling through such a modification group.
The biosubstance binding site may be appropriately adopted depending on the use of a fluorescent labeling in the targeting analysis and its embodiment is not specifically limited. For example, lectin or an antigen used for detection of cancer cells, single strand (ss) DNA for use in detection of DNA in the hybridization method and proteins such as biotin, adipin or antibodies for use in detection of proteins in the ELISA method can be adopted as the biosubstance binding site of the invention.
Meanwhile, examples of a compound forming the surface binding site include a CH3 group as one of an alkyl group, or SiC, SiOCH, SiCNH and the like, as a compound containing C having an unbonded bond; SiNH, SiCNH and the like as an amino group containing compound; and a silane coupling agent such as (COOH)—Si(OCH3)3 as an organic compound containing a carboxyl group. The foregoing carboxyl group or amino group may be introduced by allowing a compound containing such a functional group (e.g., a silane coupling agent) to bind to the nanoparticle surface or in such a manner that a compound not containing such a functional group is allowed to bind to the nanoparticle surface, followed by formation of a carboxyl or amino group through reaction.
Of the foregoing, SiOCH is a compound formed by replacing a part of the matrix of SiO2 by a methyl group and after such a methyl group is oxidized to form a carboxyl group (carboxylation reaction), a biosubstance binding site may be introduced thereto by a method using an amido-bond, as described later. Similarly, SiC and SiCNH can also introduce a biosubstance binding site through a carboxyl group.
SiNH is a compound containing an amino group formed by replacing a part of amorphous Si3N4 by a hydrogen atom. Similarly to the foregoing, a biosubstance binding site can be introduced through a non-binding bond of N or a bond via an amino group. SiCNH can also similarly introduce a biosubstance binding site through an amino group.
Such a compound, in cases when employing the photo-CVD method, exists on the shell surface of the core/shell nanoparticulate in such a form that an island portion in a so-called island/sea structure is layered on the half-face side of a spherical particulate. A compound such as SiC, SiOCH, SiNH or SiCNH and a compound forming a shell are bonded mainly through a covalent bond of Si. Accordingly, it is presumed that a strong bond with SiO2 is formed through O.
The fluorescent labeling substance may contain modification groups other than the above-described ones, for example, a modification group to enhance hydrophilicity, within a range of not inhibiting the effect of the invention.
The area of a region having no modification group on the particulate surface of the fluorescent labeling substance of the invention preferably accounts for at least 50% of the total surface area of the nanosized particulates to attain sufficient fluorescence visibility for analysis and also not to adversely affecting bonding to the targeted biosubstance. For instance, when a fluorescent labeling substance bound to an affected area (such as a cancer cell) is observed from above, if at least the upper half of a spherical nanoparticle has a light-emitting function, there is no need to have a modification group.
In the invention, “an area of the region having no modification group” refers to the area of a region which is not covered with a molecule forming the modification group and which can be measured through observation by using a TEM.
Such an area having no modification group is preferably formed continuously on the nanoparticulate surface. For example, a reaction of introducing a modification group is allowed to proceed only on half-surface of a nanoparticle by the method as described later, thereby enabling to secure a continuous region having no modification group on the opposite half-spherical side. Similarly, a modification group may be allowed to exist only on one half of the cylindrical surface of a nanorod or on one side of top and bottom parallel surfaces of the nanorod. “Cylindrical surface” and “one side of parallel surfaces” of a nanorod refer to the side surface and one of parallel surfaces of a cylindrical nanorod. A nanorod of an oval sphere is presumed to be a pseudo-spherical form and its half spherical surface is ascribed to be a half-surface of a cylindrical surface, and such a nanorod is presumed to have no parallel surface.
When such a nanoparticulate, as described above is bonded to a biosubstance [
Inorganic fluorescent nanoparticles usable in the invention can be prepared in accordance with commonly known methods. The preparation method is not specifically limited but examples thereof include gas phase processes such as a CVD method, a laser ablation method, a silane degradation method and a Si electrode vaporization method, and liquid phase processes such as an electrolysis method and a reversed micelle method. Inorganic nanoparticles prepared by these methods may be suspended in liquid or fixed on a plate, but any form is applicable so long as introduction of a modification group is feasible.
The modification group of the invention can be introduced to the nanoparticulate surface, for example, in such a manner that a compound forming a surface binding site is introduced onto the nanoparticulate surface and a material forming a biosubstance binding site is allowed to bind to this compound. Alternatively, a compound forming a spacer is allowed to bind to a compound forming a surface binding site and further thereto, a material forming a biosubstance binding site may also be allowed to bind.
Introduction of a surface binding site is not limited to a specific method but can be achieved by appropriate methods but the use of a photo-CVD method, as described below, is cited as the preferred embodiment of the invention in terms of a modification group being easily introduced to a selected region.
A core/shell nanoparticle comprised of a Si core and a SiO2 shell is prepared and the nanoparticle is fixed on the planar surface. Subsequently, light is irradiated from only one direction and one side of the nanoparticle is exposed thereto in an atmosphere of SiH(CH3)3 and N2O and is allowed to react by a photo-CVD method. Thereby, a layer composed of SiOCH, which corresponds to a layer obtained by replacing a part of a SiO2 matrix with an alkyl group, for example, CH3 (methyl group), is formed to cover half of the spherical SiO2 shell surface. Further, oxidation of CH3 under an atmosphere of CO2 converts the methyl group to a carboxyl group.
Using a core/shell nanoparticle of a Si core and a SiO2 shell, light is irradiated from only one direction and the nanoparticle is exposed thereto in an atmosphere of SiH4 and NH3 and is allowed to react by a photo-CVD method. Thereby, a layer comprised of amorphous Si3N4 containing many hydrogen atoms, that is, SiNH having NH2 (amino group) is formed to cover a part of the spherical SiO2 shell surface.
Using a core/shell nanoparticle comprised of a Si core and a SiO2 shell, light is irradiated from one direction and the nanoparticle is exposed thereto in an atmosphere of C4F8—C2H2 and is allowed to react by a photo-CVD method. Thereby, a layer having an amorphous C—H membrane, that is, CH3 (methyl group) is formed so as to cover a part of the spherical SiO2 shell surface. Similarly to the foregoing embodiment 1, oxidation of CH3 in an atmosphere of CO2 converted the methyl group to a carboxyl group.
There is prepared a core/shell nanorod comprised of a Si core and a SiO2 shell, in which a Si nanorod is vertically formed on a Si substrate through microfabrication and oxidized with O2 to form Si/SiO2. Then, etching the Si substrate side, the nanorod is separated from the substrate and heated in an atmosphere of NH3 to convert only the Si portion of the bottom surface to SiNH. Thereby, a layer comprised of SiNH and containing NH2 (amino group) is formed only on the bottom surface.
In the invention, an organic molecule, called a bi-functional cross-linker, such as SMCC (sulfomaleimidomethylcyclohexanecarboxylic acid sulfohydroxysuccinimide ester sodium salt) may be linked as a spacer.
The foregoing SMCC has two functional sites exhibiting directivity to an amino or thiol group, and one of them is allowed to link, for example, SiNH and the other one can be used for bonding to a compound to form a biosubstance binding site. Further, there can also be usable a bifunctional cross-linker having a structure in which a material to form a surface binding site and a material to form a biosubstance bonding side are introduced to both ends of an oxyalkylene, such as polyethylene glycol (PEG).
A biosubstance binding site, in which the above-described compound to form the surface binding site or a functional group capable of bonding to a functional group contained in a bifunctional cross-linker is preliminarily introduced to a part of ssDNA, adipin, biotin or an antibody by commonly known means, can be introduced to a modification group. For example, when a nanoparticle having introduced a carboxy group and lectin having introduced a carboxyl group are allowed to react, biotin is introduced to a modification group through a peptide bonding. Similarly, when a nanoparticle having introduced an amino group and lectin having introduced a carboxyl group are reacted, biotin is introduced to a modification group through peptide bonding.
The present invention will be further described with reference to examples, but the invention is by no means limited to these.
First, in accordance with the known method (JP-A No. 5-224261), core/shell nanoparticles comprised of a 2 nm diameter Si core and a 1.5 nm thick SiO2 shell were prepared by a microwave plasma decomposition method of SiH4 gas and an oxidation treatment by a strong-alkali treatment, as shown in
First, in accordance with the known method (JP-A No. 5-224261), core/shell nanoparticles comprised of a 2 nm diameter Si core and a 1.5 nm thick SiO2 shell were prepared by an oxidation treatment of a microwave plasma decomposition method of SiH4 gas and a strong-alkali treatment, as shown in
Subsequently, this amino group or unbonded bond of N was linked to a carboxy group of LECTIN through peptide linkage to obtain a fluorescent labeling substance in which the LECTIN was bonded to a part of the spherical surface, as shown in
Similarly to the treatment conditions of the photo-CVD of Example 1 as shown in
Si nanorods were prepared in accordance with the known method [as disclosed in J. Rose et al., Mat. Res. Symp. Proc. Vol. 832 (2005), F7.14.1]. First, a solution, in which surfactant Triton X-100 was dissolved in a mixture of water and methanol at a ratio of 1:400 and polystyrene of a 300 nm diameter sphere was further dissolved therein, was coated on a Si (111) substrate and allowed to stand in a desiccator for one day. Thereby, the structure of the Si substrate was covered with a polystyrene sphere monolayer. Subsequently, etching by Ar+ was performed with applying a pressure of 10.7 Pa to make the size of polystyrene spheres smaller to form a mask used for the subsequent step. As shown in
In the structure shown in
In accordance with the known method (as described in JP-A No. 5-224261), core/shell nanoparticles having a 2 nm diameter core and a 1.5 nm thick shell were prepared through a microwave plasma decomposition method of SiH4 gas and oxidation by a strong alkali treatment, as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-021391 | Jan 2006 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/323560 | 11/27/2006 | WO | 00 | 7/25/2008 |
