KR20110008400A - Nanocomplex of core-shell structrue for labeling taget molecule and method for labeling taget molecule using the same - Google Patents

Abstract

PURPOSE: A nanocomposite for labeling a target molecule of core-shell structure is provided to visually detect the presence of target material without requiring a fluorescent microscope or analyzer. CONSTITUTION: A nanocomposite for labeling a target molecule of a core-shell structure contains a core containing a plurality of metal nanoparticles or luminescent nanoparticles which cause surface plasmon resonance(SPR); and a polymer shell having one or more functional groups which selectively binds to the target molecule. The target molecule is selected from the group consisting of DNA, RNA, peptide, protein, aptamer, antigen, antibody, hapten, organic compound, and inorganic compound. A method for labeling the target material binds the nanocomposite to the target molecule.

Description

Nanocomplex of Core-Shell Structrue for Labeling Taget Molecule and Method for Labeling Taget Molecule Using the Same}

The present invention relates to a nanocomposite for labeling a target substance having a core-cell structure and a labeling method using the same.

In bioanalytical science and bioengineering, recent studies in genomics and proteomics produce more sequence data. Thus, there has been a need for new technologies that can screen large numbers of biomolecules and their ligands at the same time quickly.

In order to analyze gene expression patterns, gene defects, protein distribution, and reaction patterns in samples, analytical methods such as biochips are used. In order to determine whether a target substance capable of binding to a probe exists in a sample, There was a need for a system capable of detecting the binding of a probe immobilized on a target material to a target material.

Most DNA chips for genetic analysis now label fluorescent dyes on sample DNA, react with probes on the chip, and use a confocal microscope or CCD camera to detect fluorescent material on the chip surface. Method (US Pat. No. 61,410,96), which is difficult to miniaturize and cannot see the digitized output, much research is being conducted on the development of a new detection method that can produce an electrical signal. .

On the other hand, many research institutes including Clinical Micro Sensors are investigating a method of electrochemically detecting DNA hybridization using a metal compound that is easily oxidized / reduced (US Patent No. 6096273, 6090933). When DNA hybridizes, other compounds, including metals that are easily oxidized / reduced, complex together and are detected electrochemically (Kusakabe, T. et al., Anal . Chem ., 70: 4670). , 1998). However, the electrochemical method also had the disadvantage of requiring separate labeling. In recent years, researches on DNA detection using nanoparticles have been actively conducted. Instead of fluorescently labeled targets, hybridization is performed using targets labeled with Au nanoparticles, and then stained with silver. An analysis (Mirkin et al., Science , 289: 1757, 2000) or an electrical detection study (Mirkin et al., Science , 295: 1503, 2002) is a good example. However, if the concentration of the target material in the sample is too low, the methods were not suitable for detecting the presence of the target material in the sample. Therefore, the development of a labeling method with improved efficiency that can effectively detect even a low concentration of the target material in the sample was required. In addition, conventional nanoparticles have been used for imaging in combination with the antibody targeting the target material, there was a problem that the surface characteristics of the nanoparticles to be adjusted to attach the nanoparticles to the antibody.

Therefore, the present inventors can effectively detect the target substance present in the trace amount in the sample effectively, and as a result of a diligent effort to develop a new target substance labeling method capable of visually detecting the presence of the target substance without a separate device, a new core -The nanocomposite of the cell structure was prepared, and it was confirmed that the trace amount of the substance can be effectively detected by binding and labeling the target substance, thereby completing the present invention.

It is an object of the present invention to provide a labeling material for a new target material and a labeling method using the same that can maximize the detection efficiency that can also detect a trace amount of a biological material or compound.

In order to achieve the above object, the present invention includes a core containing a plurality of metal nanoparticles or light emitting nanoparticles (core) that can cause Surface Plasmon Resonance (SPR); And

Provided is a nanocomposite for labeling a target material including a polymer polymer shell having one or more functional groups capable of selectively binding to a target material.

The present invention is also characterized in that the micro-emulsion is formed by adding a metal nanoparticle or a light emitting nanoparticle-containing organic solution capable of generating a plurality of Surface Plasmon Resonance (SPR) to a polar polymer-containing polar solution. Provided is a method of preparing a nanocomposite for substance labeling.

The present invention also provides a method for labeling a target material, characterized in that for bonding the nanocomposite to the target material.

The present invention also provides a composition for labeling a target substance, comprising the nanocomposite.

The present invention has an effect of providing a nanocomposite for labeling a target material and a labeling method using the same. The nanocomposite according to the present invention enables specific detection of even a very small amount of target material, and concentrates color or light emission to visually detect the presence or absence of a target material without a separate fluorescence microscope or analysis device. By amplification, it can be applied to various parts such as detection of biological substances, discovery of medicine, etc., which is very useful.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

As used herein, a "core-shell" structure refers to a structure in which an inorganic nanomaterial or an inorganic-organic hybrid material exists inside as a core, and a polymer polymer surrounds the core as an outer shell, that is, a shell. it means.

As used herein, the term "surface plasmon resonance" (SPR) is a photoelectron effect that occurs in a metal such as gold, and when light of a specific wavelength is irradiated to the metal, a resonance phenomenon occurs in which most light energy is transferred to free electrons. As a result, the phenomenon that occurs when surface waves occur is called SPR. The wavelength of surface plasmon absorption varies with the type of metal, the size of the metal particles, the coating, and the dielectric constant of the coating material. The position of the surface plasmon absorption peak can be predicted by the well-known Mie resonance condition. have.

In the present specification, the term "superparamagnetic material" means that the magnetization disappears by thermal motion when no external magnetic field is applied, whereas the degree of magnetization induced when an external magnetic field is applied is much larger than that of paramagnetic material. It is a substance that exhibits self-saturation. In general, when the ferromagnetic material is reduced to the size of the terminal sphere, the phase change into a superparamagnetic body, the size of the terminal sphere is reported to have a diameter of about several nanometers to several hundred nanometers, although the size varies depending on the material. As used herein, the term “superparamagnetic nanoparticles” includes nanoparticles that are finely broken into nano-sizes and have superparamagnetic properties.

The present invention relates to a nanocomposite having a new structure with improved labeling efficiency to specifically detect a trace amount of a target material, and the present invention provides a metal capable of generating surface plasmon resonance (SPR). A core containing a plurality of nanoparticles or light emitting nanoparticles; And a polymer polymer shell having at least one functional group capable of selectively binding to a target material. Figure 1 (a) is a schematic diagram of one embodiment of a nanocomposite according to the present invention.

The nanocomposite according to the present invention is characterized in that it has a diameter of 20 ~ 500nm, more preferably 50 ~ 100nm, wherein the particle size is less than 20nm, it is difficult to contain a plurality of nanoparticles, if more than 500nm in aqueous solution There is a problem that is difficult to distribute.

In the present invention, the target material for the purpose of labeling may include DNA, RNA, peptide, protein, antigen, antibody, hapten, organic compound or inorganic compound, but is not limited thereto. In this case, in addition to the analyte for detection, DNA, RNA, peptide, protein, antibody, aptamer, etc. used for detecting the analyte may also be a target material for labeling.

The present invention is characterized by integrating a plurality of nanoparticles, that is, several tens or thousands, preferably tens or hundreds of nanoparticles, into a single cell so that even a very low concentration of the target substance can be specifically detected by the naked eye. . At this time, the nanoparticles constituting the core to be integrated into the cell uses metal nanoparticles or light emitting nanoparticles that can cause SPR.

Metal nanoparticles that can cause the SPR is Au, Ag, Pt, Pd, Ir, Rh, Ru, Al, Cu, Te, Bi, Pb, Fe, Ce, Mo, Nb, W, Sb, Sn, V, Any one or more metals selected from the group consisting of Mn, Ni, Co, Zn and Ti may be used. Among them, Ag shows the sharpest SPR resonance peak, and Au shows excellent stability, which is preferable for the label according to the present invention. In particular, gold (Au) nanoparticles have different maximum absorption wavelengths in the absorption spectrum depending on the particle size when dispersed in a solution. For example, when gold nanoparticles with a diameter of about 13 nm are well dispersed in aqueous solution, the maximum absorption wavelength is about 530 nm and the solution becomes reddish. However, when the particles are aggregated and aggregated, the maximum absorption wavelength shifts to the long wavelength and the color of the solution becomes bluish. On the other hand, Ag is known to have 4 times larger SPR signal than Au when it is the same size, and when aggregated, it has a dark gray color. Therefore, by agglomerating the polymer into the polymer cell, the visible color is obtained. By binding the nanocomposite containing the core to the target material, it is possible to detect a trace amount of a biological material.

 At this time, the metal is a metal that can cause surface plasmon resonance, due to the combination thereof will cause a change in the surface plasmon resonance spectrum. Therefore, this can be measured using a spectrum analyzer. At this time, by using a plurality of gold (Au) coated superparamagnetic nanoparticles, it is possible to check the visible color change (blue light) according to the aggregation of the gold nanoparticles as described above without a separate spectrum analyzer. In addition, in the case of silver (Ag), when a plurality of silver nanoparticles are aggregated, the gray color may be visually confirmed. At this time, more preferably, the superparamagnetic nanoparticles are coated with gold (Au) having high surface stability, and then reduced to Ag having the sharpest SPR resonance peak and double coated with Au and Ag to improve the efficiency of SPR spectrum analysis. Can be. In addition, it can be seen that the color change according to the aggregaton becomes more intense (black color) during the double coating.

On the other hand, by measuring the electrochemical signal in addition to the spectrum or color change measurement, it is possible to detect the presence of the target material. Those skilled in the art having ordinary skill in the art, when the core of the nanocomposite according to the present invention is composed of metal nanoparticles capable of generating a plurality of SPR, commonly known electrochemical signals for measuring electrochemical signals Signal measurement method can be performed. That is, a current that is not located between the electrodes and does not flow between the electrodes flows through the metal nanoparticles, preferably, Au particles, thereby detecting an electrical signal or using an electrochemical signal using a catalytic reaction of the metal nanoparticles. Can be detected. Therefore, if the detection device capable of measuring the signal by the SPR, it will be obvious that it can be used for the identification of the nanocomposite for labeling the target material according to the present invention.

In addition, the metal nanoparticles capable of generating SPR may be preferably characterized in that the metal-coated dielectric nanoparticles (dielectric nanoparticles) capable of generating SPR. It has a dielectric core (dielectric core) therein, but may be used as a dielectric core, such as silica, iron oxide, Hydroxy Apatite polymer, preferably gold (Au) coated silica nanoparticles.

When labeled with the metal-coated dielectric nanoparticles (SPR) that can cause the SPR as described above, by confirming the visible color due to the aggregation of the metal, or by measuring the signal using an electrochemical signal detection device, The nanocomposite for labeling the target material according to the invention can be confirmed.

On the other hand, the light emitting nanoparticles may be characterized in that the nanostructure in which a plurality of organic light emitting compounds are bonded, more specifically, a metal oxide in which silica nanoparticles or organic fluorescence particle molecules on which fluorescent molecules are loaded (eg, For example, rare earth oxide) nanoplate particles.

In the case of light emitting nanoparticles, it may be characterized in that it is preferably loaded or accumulated several hundred to tens of thousands of fluorescent molecules, the organic fluorescent material that can be used is not limited, in particular, Rhodamine B isothiocyanate (Rhodamine B Isothiocyanate (RITC) or Fluoresceine isothiocyanate (FITC) is preferably used, but any organic fluorescent material generally used may be used without limitation. In addition, Alexa Fluor, Rhodamine Red-X, Texas Red, Tetramethylrhodamine, Cascade Blue, DAPI (4 ', 6) Chemically modified organic fluorescent materials such as -diamidino-2-phenylindole), coumarines or Lucifer Yellow can also be used.

At this time, the integration of the fluorescent molecules into the metal oxide nanoparticles (nanoplate) can be carried out by a generally known fixing method, for example, on the surface of the metal oxide nanoparticles through a functional group capable of strongly binding to the surface of the metal oxide nanoparticles. It may be immobilized in ligand form. In this case, the functional group may be an aldehyde group, an amine group, a thiol group, a sulfide group, a disulfide group, a silane group, a carboxyl group, an alcohol group, an epoxy group, a thiol group, an alkyl halide group, an alkyl group, an alkene group, an alkyne group, an aryl group, or the like. Combinations may be used. First, the functional groups are attached to fluorescent molecules such as organic fluorescent molecules, and then the fluorescent molecules are fixed to ligands on the surface of the metal oxide nanoparticles.

The integration of tens to tens of thousands of light emitting nanoparticles into the cores provides a strong fluorescence intensity, so that even a low concentration of the target material can be easily detected. The signal of the light emitting nanoparticles may be measured using a fluorimeter or a fluorescence microscope according to a conventional method.

On the other hand, the core containing a plurality of nanoparticles as described above is surrounded by a polymer polymer shell (shell) having one or more functional groups capable of selectively binding to the target material, wherein the polymer polymer is preferably a parent By using an amphiphilic block copolymer (amphiphilic block copolymer) can be easily prepared during the preparation in a microemulsion method to increase the economics, the prepared nanocomposite can be very excellent in dispersibility of the aqueous solution.

The binding of the nanocomposite to the target material according to the present invention is performed by one or more functional groups on the cell of the nanocomposite, but the functional group is not limited thereto, and preferably, a functional group capable of selectively binding to the target material. Is a thiol group, a sulfide group, a disulfide group, a silane group, a carboxyl group, an amine group, an alcohol group, an aldehyde group, an epoxy group, a thiol group, an alkyl halide group, an alkyl group, an alkene group, an alkyne group, an aryl group, or a combination of two or more of these functional groups. Can be used. Such functional groups may be present in plural on the surface of the nanocomposite, but are not limited thereto, and there may be several to several thousand on the surface of the nanocomposite.

In the present invention, additionally superparamagnetic nanoparticles may be included in the core or attached to the shell of the cell to facilitate binding of the nanocomposite according to the present invention to the target. The superparamagnetic nanoparticles may be made of a metal selected from the group consisting of Iron-Oxide, Iron-Cobalt, Iron-Nickel and transition metals, preferably γ-Fe ₂ O ₃ or Fe ₃ O ₄ It is available.

The nanocomposite for labeling the target material according to the present invention as described above may be prepared by a microemulsion method, but the prepared nanocomposite generally has a spherical shape, but is not limited thereto. For example, rather than completely round, it may be of an oval or tubular-like shape which is desirable in many applications. The sheath may be of any thickness compatible with the method of making the present nanocomposites, but relatively thin sheaths are generally preferred for use of the invention for labels.

Nanocomposites according to the present invention may be prepared by a method that specifically comprises the following steps:

(a) mixing metal nanoparticles or luminescent nanoparticles that can cause a plurality of Surface Plasmon Resonance (SPR) into an organic solvent;

(b) adding the nanoparticle-containing organic solution to an amphiphilic block copolymer aqueous solution;

(c) stirring the mixed solution to form a microemulsion to form a nano-composite having a core-cell structure; And

(d) modifying the nanocomposite to have one or more functional groups for binding to a target material.

First, metal nanoparticles or light emitting nanoparticles that cause SPR are mixed on an organic solvent. The organic solvent capable of dispersing the nanoparticles in the colloidal phase is not particularly limited as long as it has a weak polarity. For example, dichloromethane, toluene, Anisole, chloroform or dichlorobenzene can be used.

Then, the nanoparticle-containing organic solution is added to the amphiphilic block copolymer aqueous solution, and the mixed solution is stirred to form a core-cell structure. Solutions containing amphiphilic block copolymers utilize polar solvents, which are inexpensive and readily available for water, nontoxic, easy to handle and store, are compatible with many other precipitation reactions, and can be mixed with many nonpolar solvents. It is preferable to use it since it cannot be.

Conventional microemulsion methods require surfactants to reduce surface tension and form microemulsions at the interface of polar and nonpolar liquids, but the present invention uses an amphiphilic block copolymer and does not require a separate surfactant. . In addition, it can be easily prepared during the preparation by the microemulsion method to increase the economics, and the prepared nanocomposite was very excellent in dispersibility of the aqueous solution.

An amphiphilic block copolymer is an embodiment consisting of only one segment A and one segment B (diblock copolymer (AB) or one consisting of segment A and two segments B bonded to both ends thereof (triblock Copolymer BAB) or a complex structure in which several segments B are attached to one segment A [of course, it may further contain one or two terminal segments B; composite block copolymer A (-B r], wherein segment A and segment B are as described above, r has a value greater than 2, segment A and segment B may be connected by direct bond or by the aforementioned bridging member, segment A, Preferably some or all of the segments B may have crosslinkable groups, in particular crosslinkable CC double bonds.

In another embodiment, the amphiphilic block copolymer of the present invention consists of one segment B and two segments A bonded at both ends thereof (triblock copolymer ABA), or several segments A in one segment B The complex structure to which it is attached [of course, it may further contain one or two terminal segments A; Complex block copolymer B (-A) r].

These copolymers are for example the diblock, triblock or complex block copolymers AB, BAB or A (-B) _r described above where segment A and segment B are, for example, carbonyl, carbonate, ester , By an amide, urea or urethane functional group, or in particular a crosslinker -XR ^* -X-, wherein X is the aforementioned functional group, and R ^* is, for example, divalent aliphatic having up to 20 carbon atoms, Alicyclic, aromatic or araliphatic groups (e.g., straight or branched C6-C10-alkylene); cyclohexylene-methylene or cyclohexylene-methylene-cyclohexyl, each unsubstituted or substituted by one to three methyl groups Or phenylene or phenylene-methylene-phenylene unsubstituted or substituted with methyl]. Particularly preferred copolymers are -NH-C (O) -NH-R ^* -NH-C. (O) -NH- or -OC (O) -NH-R ^* -NH-C (O) -O-, where R ^* is as defined above Is the same).

As described above, the nanocomposite formed by using the microemulsion method may be modified to have one or more functional groups for binding to a target material, and the functional group is not limited thereto, but preferably may selectively bind to the target material. The functional group which may be thiol group, sulfide group, disulfide group, silane group, carboxyl group, amine group, alcohol group, aldehyde group, epoxy group, thiol group, alkyl halide group, alkyl group, alkene group, alkyne group, aryl group or two or more of these Combinations of functional groups can be used.

The binding of the nanocomposite to the target material according to the present invention is performed by one or more functional groups on the cell of the nanocomposite, which is directly bound to the target material or specific to the target material such as DNA, RNA, protein, aptamer and antibody. A conjugate capable of binding can be linked to the functional group to label and detect the analyte (FIG. 1 (b)).

Accordingly, in another aspect, the present invention provides a composition for labeling a target material comprising the nanocomposite and a method for labeling the target material, wherein the nanocomposite is bonded to the target material.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is an exemplary view of a target material labeling nanocomposite (a) and labeling method (b) according to the present invention.

Claims (23)

A core containing a plurality of metal nanoparticles or luminescent nanoparticles capable of producing Surface Plasmon Resonance (SPR); And A nanocomposite for labeling a target material comprising a polymer polymer shell having one or more functional groups capable of selectively binding to a target material. The nanocomposite of claim 1, wherein the target material is selected from the group consisting of DNA, RNA, peptide, protein, aptamer, antigen, antibody, hapten, organic compound and inorganic compound. . The nanocomposite of claim 1, wherein the metal nanoparticles are metal coated dielectric nanoparticles. 4. The nanocomposite of claim 3, wherein the metal-coated dielectric nanoparticles are Au coated silica nanoparticles. The method of claim 1, wherein the metal capable of causing surface plasmon resonance is Au, Ag, Pt, Pd, Ir, Rh, Ru, Al, Cu, Te, Bi, Pb, Fe, Ce, Mo, Nb, W, Sb, Sn, V, Mn, Ni, Co, Zn and Ti is any one or more metal selected from the group consisting of nanocomposite for labeling the target material. The nanocomposite of claim 5, wherein the metal is Au or Ag. The nanocomposite of claim 1, wherein the metal nanoparticles are double coated with Au and Ag. The nanocomposite of claim 1, wherein the light emitting nanoparticles are nanostructures in which a plurality of light emitting organic compounds are bonded. The nanocomposite of claim 1, wherein the light emitting nanoparticles are silica nanoparticles on which fluorescent molecules are loaded or metal oxide nanoplate particles on which organic fluorescent particle molecules are integrated. The method of claim 1, wherein the functional group capable of selectively binding to the target material is a thiol group, sulfide group, disulfide group, silane group, carboxyl group, amine group, alcohol group, aldehyde group, epoxy group, thiol group, alkyl halide group, alkyl group , Alkene group, alkyne group, aryl group or a combination of two or more of these functional groups of the target material labeling nanocomposite. The nanocomposite of claim 1, wherein the polymer is an amphiphilic block copolymer. The nanocomposite of claim 1, wherein the superparamagnetic nanoparticles are included in the core or attached to the shell of the cell. The nanocomposite of claim 12, wherein the superparamagnetic nanoparticles are made of a metal selected from the group consisting of Iron-Oxide, Iron-Cobalt, Iron- Nickel, and transition metals. The nanocomposite of claim 13, wherein the superparamagnetic nanoparticles are γ-Fe ₂ O ₃ or Fe ₃ O ₄ . The nanocomposite of claim 1, wherein the particle size of the nanocomposite is 50-100 nm. Claim 1 to 14, characterized in that to form a microemulsion by adding an organic solution containing metal nanoparticles or light-emitting nanoparticles that can cause a plurality of Surface Plasmon Resonance (SPR) to a polar solution containing a polymer polymer; Method for producing a nanocomposite for labeling any one of the target materials 17. The method of claim 16, comprising the following steps: (a) mixing metal nanoparticles or luminescent nanoparticles that can cause a plurality of Surface Plasmon Resonance (SPR) into an organic solvent; (b) adding the nanoparticle-containing organic solution to an amphiphilic block copolymer aqueous solution; (c) stirring the mixed solution to form a microemulsion to form a nano-composite having a core-cell structure; And (d) modifying the nanocomposite to have one or more functional groups for binding to a target material. 18. The method of claim 17, wherein the superparamagnetic nanoparticles are mixed together in the organic solvent of step (a). 18. The method of claim 17, wherein the superparamagnetic nanoparticles are attached to the shell of the nanocomposite of step (c). A method for labeling a target material, characterized in that to bind the nanocomposite of any one of claims 1 to 15 to the target material. A composition for labeling a target substance, comprising the nanocomposite of any one of claims 1 to 15. The composition of claim 21, wherein the binder specifically binding to the target material is linked to a functional group of the nanocomposite. The composition of claim 22, wherein the conjugate capable of specifically binding to the target material is selected from the group consisting of DNA, RNA, peptide, protein, aptamer, and antibody.

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