JP2008260724A - Ferromagnetic nanoparticles having organic molecule immobilized thereon, method for producing the same and method for separating the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide ferromagnetic nanoparticles modified with an organic molecule, which can be subjected to magnetic separation at a nano-size at room temperature. <P>SOLUTION: The ferromagnetic nanoparticles having an organic molecule immobilized thereon are prepared by immobilizing an organic molecule on ferromagnetic FePt nanoparticles. The method for producing the ferromagnetic nanoparticles includes treating the ferromagnetic FePt nanoparticles with a fatty acid and an aliphatic amine to immobilize the fatty acid and the aliphatic amine on the ferromagnetic FePt nanoparticles, and treating the resultant nanoparticles with an organic compound having a catechol residue and an organic compound having a mercapto group. The method for separating the ferromagnetic nanoparticles comprises separating the ferromagnetic nanoparticles and/or particles prepared by binding another material to the ferromagnetic nanoparticles by magnetism from a solution containing the nanoparticles and/or particles prepared by binding other materials to the ferromagnetic nanoparticles. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a ferromagnetic nanoparticle modified with an organic molecule, and a production method and a separation method thereof.

  In recent years, nanoparticles have been expected to have a wide range of applications in biological fields such as biological separation, various measurements, and drug delivery systems. The characteristics of the nanoparticles are expected to be expanded applications and biocompatibility due to the decrease in volume due to the nanoparticle size, and increased recognition ability and shortened recognition time due to the increase in specific surface area. Therefore, the development of nanoparticles has become a major theme, and in particular, magnetic nanoparticles using nanoparticles as magnetic materials can be applied in fields that utilize their magnetic properties, such as measurement by MRI and construction of drug delivery systems by magnetic induction. Is expected (Non-patent Document 1).

  However, as represented by the present iron oxide nanoparticles, when the volume of the magnetic particles is reduced, the magnetic moment becomes disordered by the thermal energy at room temperature, and its magnetic properties are lost. Magnetic separation at nano-size has been considered impossible.

  Generally, when the product of the magnetic anisotropy energy Ku and the volume V of the magnetic material is less than the thermal energy KT, the properties as the magnetic material are lost. It is essential to have a high Ku.

Applied Physics Vol.72, No.7, 909 (2003)

  An object of the present invention is to provide a ferromagnetic nanoparticle modified with an organic molecule that can be magnetically separated in nanosize at room temperature.

The gist of the present invention is as follows.
(1) Organic molecule-immobilized ferromagnetic nanoparticles obtained by immobilizing organic molecules on ferromagnetic FePt nanoparticles.
(2) an organic molecule immobilized ferromagnetic nanoparticles according to (1) which ferromagnetic FePt nanoparticles have an L1 ₀ ordered structure.
(3) The organic molecular immobilization strength according to (1) or (2) above, wherein the ferromagnetic FePt nanoparticles are obtained by treating an Fe complex and a Pt complex with a polyol solvent having a boiling point of 300 ° C. or higher. Magnetic nanoparticles.
(4) The organic molecule-immobilized ferromagnetic nanoparticle according to any one of (1) to (3), wherein the organic molecule immobilized on the ferromagnetic FePt nanoparticle is selected from a specific binding partner and a physiologically active substance. particle.
(5) After treating the ferromagnetic FePt nanoparticles with a fatty acid and an aliphatic amine to immobilize the fatty acid and the aliphatic amine on the ferromagnetic FePt nanoparticles, an organic compound having a catechol residue and an organic having a mercapto group The method for producing an organic molecule-immobilized ferromagnetic nanoparticle according to any one of the above (1) to (4), which is treated with a compound.
(6) The production method according to (5), wherein at least one of the organic compound having a catechol residue and the organic compound having a mercapto group has a residue selected from a specific binding partner, DNA, an enzyme, and a physiologically active substance. .
(7) Organic molecule-immobilized ferromagnetic nanoparticles obtained by the production method according to (5) or (6).
(8) Organic molecule-immobilized ferromagnetic nanoparticles according to any one of (1) to (4) and (7), and / or particles obtained by binding other substances to the organic molecule-immobilized ferromagnetic nanoparticles Organic molecule-immobilized ferromagnet, wherein the organic molecule-immobilized ferromagnetic nanoparticle and / or particles having other substances bonded to the organic molecule-immobilized ferromagnetic nanoparticle are separated from the solution comprising Nanoparticle separation method.

  ADVANTAGE OF THE INVENTION According to this invention, the ferromagnetic nanoparticle modified with the organic molecule which can be magnetic-separated by nanosize at room temperature can be provided.

  The ferromagnetic FePt nanoparticle used in the present invention is composed mainly of Fe atoms and Pt atoms (usually, 50 mol% or more, preferably 90 mol% or more in total of Fe atoms and Pt atoms in all metal atoms). A particle having a particle diameter of about 10 nm and showing magnetic hysteresis at room temperature.

L1 ₀ has a high crystal magnetic anisotropy in FePt alloy exhibiting regular structure (International Center For Diffraction Data, Powder Diffraction File SET43 Inorganic and organic, 43-1539). This high magnetocrystalline anisotropy is considered to be possible in principle for ultra-high density recording of 1 Tbit / inch ² or more (K. Inomata, T. Sawa, S. Hashimoto, J. Appl. Phys. 1996, 64, 2537), and is expected to be applied to future high-density recording media.

  In the present invention, FePt nanoparticles exhibiting nano-order ferromagnetism are used because they have high magnetocrystalline anisotropy energy Ku.

FePt alloy may take two structural an fcc structure is a L1 ₀ ordered structure and metastable structure is stable phase. Here L1 ₀ ordered structure has the axis of easy magnetization in the c-axis, while exhibiting ferromagnetism because it has a high magnetocrystalline anisotropy, fcc structure is a metastable structure is absent easy magnetization axis, It shows paramagnetism (FIG. 1).

Hereinafter, the origin of the high magnetocrystalline anisotropy of the L1 ₀ -FePt alloy will be described.
The 4d transition metal Pd and the 5d transition metal Pt are called platinum group atoms and show paramagnetism in the bulk simple substance, but are known to be metals immediately before the ferromagnet. Further, it is known that this platinum group exhibits a large spin polarization by alloying with the iron group.

FIG. 2 shows a state density diagram of the FePt alloy (A. Sakuma, J. Phys. Soc. Jpn. 1994, 63, 3053).
The majority spin band of Fe and Pt has an overlap due to the hybridization of Fe3d band and Pt4d band in the vicinity of Fermi energy Ef. This hybridization induces a spin magnetic moment of Pt, and it is said that the orbital magnetic moment of Fe that has disappeared due to degeneracy is restored. Table 1 shows the spin orbit coupling energy ζ, the spin magnetic moment S, the orbital magnetic moment L, the synthesized magnetic moment M _total , and the measured magnetic moment M _ex of Fe and Pt. The induction of the magnetic moment of Pt due to the alloying of Fe and Pt can be confirmed. From the large spin orbit coupling constant induced by the induction of the magnetic moment of Pt, it can be seen that the FePt alloy has a large spin orbit interaction and that it is the origin of high magnetic anisotropy.

FePt alloys when generally Fe composition is 45% to 64% at a molar ratio, having an L1 ₀ ordered structure. However, when an FePt alloy is produced by chemical synthesis or the like, an fcc irregular phase that is a metastable phase is often taken. 2000, S.M. Sun et al. Reported a method for synthesizing FePt nanoparticles with almost no particle size distribution by a chemical method called polyol process (S. Sun, CB Murray, D. Weller, L. Folks, A. Moser, Science 2000, 287). , 1989; S. Sun, Adv. Mater. 2006, 18, 393), research on FePt alloys was accelerated, and it was expected to be practically used for magnetic recording devices. However, ordered by synthesized FePt nanoparticles also take the disordered phase is still metastable phase, heat treatment at about 600 ° C. The structural transition of the L1 ₀ structure having high magnetic anisotropy by the polyol process A process was needed. This heat treatment process causes sintering and agglomeration of FePt nanoparticles, which is a big barrier to practical use. In order to solve such problems, attempts have been made to reduce the ordering temperature by this heat treatment or prevent sintering during the heat treatment, and several methods will be exemplified below.

(1) Liquid layer direct synthesis using polyol solvents such as high-boiling ethylene glycol solvents (K. Sato, B. Jeyadevan, K. Tohji, J. Magn. Magn. Mater. 2005, 289, 1; R. Minami, Y. Kitamoto, T. Chikata, S. Kato, Electrochimica Acta 2005, 51, 864)
This technique is a technique for reducing precursors (complexes, salts, etc.) of Fe and Pt compounds using a polyol solvent as a reducing agent and a solvent. It is known that polyol solvents such as ethylene glycol solvents lower the ordering temperature to 300 ° C. or lower by reducing the reaction rate during the reduction of Fe and Pt. Furthermore, a kind of polyol solvents, 300 raised ℃ more high boiling polyol solvents tetraethylene glycol or the like having a boiling point in the liquid layer by using as the solvent a reducing and ordering at the same time, L1 ₀ ordered phase directly It is a technique to synthesize. In this approach, L1 ₀ not enough to complete ordering to a structure, although there is a disadvantage that remains in partial ordering are high magnetic anisotropy in the resulting FePt alloy confirmation, effective technique one of.

  Moreover, if this method is used, the process of annealing at 600 ° C. can be omitted, and further, synthesis in a liquid phase is possible, and thus there is an advantage that chemical surface modification after synthesis is possible. This is a great advantage for the present invention, and this is the synthesis method employed in the examples described later.

  The precursor (complex, salt, etc.) of Fe and Pt compound used here is not limited as long as it is a complex or metal salt containing Fe element and Pt element. For example, acetylacetonate (acac) complex, carbonyl complex, ethoxide compound And chloride.

  The polyol solvent used here is not limited as long as it has a boiling point of 300 ° C. or higher, but preferably tetraethylene glycol, hexaethylene glycol, octaethylene glycol, or a mixture of a high boiling organic solvent and an ethylene glycol solvent, etc. And high boiling point ethylene glycol solvents.

  The ratio of Fe complex to Pt complex is preferably 45:55 to 64:36 in molar ratio. The amount of the polyol solvent used is preferably about 100 ml per 1 mmol of metal ions.

(2) Reduction of the ordering temperature by adding a third element (JW Harrell, DE Nikles, SS Kang, XC Sun, Z. Jia, J. Magn. Soc. Jpn. 2004, 28, 847; O. Kitamura, Y Shimada, K. Oikawa, H. Daimon, K. Fukamichi, Appl. Phys. Lett. 2002, 80, 2147; T. Maeda, T. Kai, A. Kikitu, T. Nagase, J. Akiyama, Appl. Phys Lett. 2001, 78, 1104; KM Park, KH Na, JG Na, PW Jang, HJ Kim, SR Lee, IEEE Trans. Magn. 2000, 38, 1961)
This technique is a technique for increasing the atomic diffusion and promoting ordering by adding a third element in addition to Fe and Pt. Ag, Au, reports have been made that the structure transition temperature to L1 ₀ ordered phase by adding an element such Cu is decreased. Among these, Cu has a particularly large reduction in ordering temperature. In Ag and Au, the ordering temperature decreases to 400 ° C., whereas the addition of Cu decreases the ordering temperature to 300 ° C. In Ag and Au, precipitation of elements after heat treatment is observed, but not in Cu. In other words, while Au and Ag promote the diffusion of atoms by precipitation and lower the ordering temperature, Cu dissolves in the FePt alloy, thereby lowering the potential energy of the ordered phase and promoting ordering. That is why.

In the present invention, this method can also be used. Furthermore, a SiO ₂ nanoreactor method can also be used.

FePt alloy as described above can take two kinds of structure of fcc disordered phase is L1 ₀ ordered phase and the metastable phase is a stable phase. Therefore, when using an FePt alloy, it is very important to prove the structural transition and to know the order parameter indicating the proportion of the structural transition. As a method for knowing these, analysis of a diffraction pattern by X-rays is extremely effective. In the L1 ₀ ordered phase, in addition to the diffraction lines of the fcc structure which is an irregular phase, extra diffraction lines due to the regular arrangement appear. This extra diffraction line is called superlattice reflection. The appearance of the superlattice reflection originates from the fact that the extinction rule is different due to the regularization of atomic arrangement.

で与えられることからも分かるように、ｆｃｃ構造においてはｚ＝１、ｚ＝０の面からの回折線の位相とｚ＝１／２の面からの回折線の位相は半波長分だけずれるため、回折線は打ち消しあって（００１）面からのブラッグ反射は起こらない。一方、規則相であるＬ１_０構造の消滅則は、 The extinction law of the fcc structure which is an irregular phase is

As can be seen from the above, in the fcc structure, the phase of the diffraction line from the z = 1, z = 0 plane and the phase of the diffraction line from the z = 1/2 plane are shifted by a half wavelength. The diffraction lines cancel each other and Bragg reflection from the (001) plane does not occur. On the other hand, extinction rule of L1 ₀ structure is ordered phase,

で与えられる。この消滅則からも分かるようにｚ＝０、ｚ＝１の面とｚ＝１／２の面では散乱因子が異なっており、回折線は完全には打ち消しあわず、結果として超格子反射が観測される。更に、このような（００１）面などからの超格子反射の出現に加え、正方晶への変化に伴う（００２）、（２００）面からの回折線の分裂が生じる。

Given in. As can be seen from this annihilation rule, the scattering factor is different between the z = 0, z = 1 and z = 1/2 planes, and the diffraction lines do not completely cancel out. As a result, superlattice reflection is observed. Is done. Further, in addition to the appearance of superlattice reflection from the (001) plane and the like, splitting of diffraction lines from the (002) and (200) planes occurs with the change to tetragonal crystal.

  Therefore, it is possible to prove structural transition by analyzing the X-ray diffraction pattern and observing the appearance of superlattice reflection.

  In the present invention, the organic molecule immobilized on the ferromagnetic FePt nanoparticles may be appropriately selected according to the purpose, and examples thereof include a specific binding partner, DNA, enzyme, and physiologically active substance. Further, in addition to the organic molecules, depending on the purpose, for example, a polyethylene glycol chain, an alkyl chain having an ionic group, etc. for the purpose of imparting hydrophilicity, biocompatibility, dispersibility, lyophilicity to various solvents, etc. It can also be fixed in combination.

  The specific binding partner is a member of a pair of molecules that interact by specific non-covalent interactions depending on the three-dimensional structure of the molecule involved, and is a typical pair of the specific binding partner. For example, antigen-antibody, hapten-antibody, hormone-receptor, nucleic acid chain-complementary nucleic acid chain, substrate-enzyme, substrate analog-enzyme, inhibitor-enzyme, carbohydrate-lectin, biotin-avidin and virus-cell receptor. Can be mentioned.

Examples of the physiologically active substance include drugs.
For example, ferromagnetic nanoparticles with immobilized antibodies can be used for cell separation and detection / recovery of chemical substances, and ferromagnetic nanoparticles with immobilized DNA strands can be used for SNP detection, genetic diagnosis, and breed identification. The enzyme-immobilized ferromagnetic nanoparticles can be used for enzyme reaction sensing / recovery and reuse. Biotin-immobilized ferromagnetic nanoparticles can be used for various measurement methods or various substances via biotin-avidin bonds. The ferromagnetic nanoparticles having a drug immobilized thereon can be used in a drug delivery system.

  In order to immobilize organic molecules on the ferromagnetic FePt nanoparticles, the ligand containing the target organic molecule is terminated with a carboxyl group, amino group, mercapto group, hydroxyl group, etc. having high affinity for Fe and Pt. Must stand. In addition, when applied in vivo as a biomagnetic nanoparticle, a polyethylene glycol chain (PEG) (PEG) (as a spacer for imparting hydrophilicity, biocompatibility, and avoiding steric congestion between target organic molecules. Preferably, the degree of polymerization 10-20) is included in the ligand.

  Therefore, in the examples described later, two ligands, biotin-PEG-SH having biotin and mercapto groups at both ends of PEG, and dopamine-polyethylene glycol monomethyl ether (mPEG) having dopamine groups at one end are designed. -Synthesized. Here, biotin-PEG-SH is coordinated to Pt, and dopamine-mPEG is coordinated to Fe. Although dopamine-mPEG does not have a biotin group, it is intended to improve hydrophilicity and simultaneously prevents oxidation of Fe on the particle surface.

  Next, the synthesized ligand is immobilized on the ferromagnetic FePt nanoparticles, but when the ferromagnetic FePt nanoparticles are synthesized by liquid layer direct synthesis using the above-mentioned polyol solvent, the ferromagnetic material immediately after the synthesis is obtained. Since FePt nanoparticles are present in a large amount of polyol-based solvents, it is difficult to directly immobilize a synthesized ligand with a very small amount of production, so once another organic ligand such as octanoic acid is used. C6-C20 fatty acid such as oleylamine and the like and C6-C20 aliphatic amine such as oleylamine are immobilized on FePt nanoparticles, and a ligand synthesized by using a ligand exchange reaction, for example, catechol An organic compound having a residue (for example, dopamine-mPEG) and an organic compound having a mercapto group (for example, biotin-PEG-SH) are immobilized on FePt nanoparticles. Doo is preferred (Rui Hong, Nicholas O. Fischer, Todd Emrick, Vincent M. Rotello, chem. Mater, 2005, 17, 4617).

The use amount of the fatty acid is preferably 10 times or more (molar ratio) of the number of surface atoms of the target particle, and the use amount of the aliphatic amine is preferably 10 of the surface atom number of the target particle. The amount of the target synthesized ligand is preferably 10 times or more (molar ratio) of the number of surface atoms of the target particle.
The organic molecule-immobilized ferromagnetic nanoparticles of the present invention can be magnetically separated. Accordingly, the organic molecule-immobilized ferromagnetic nanoparticle of the present invention and / or the organic molecule-immobilized ferromagnetic nanoparticle by magnetism from a solution containing particles in which another substance is bonded to the organic molecule-immobilized ferromagnetic nanoparticle. And / or can be applied to a method of separating particles in which another substance is bonded to the organic molecule-immobilized ferromagnetic nanoparticles.

  For example, when using a ferromagnetic nanoparticle on which a specific antibody is immobilized as a diagnostic agent, if a specific antigen is present in the specimen, the antigen is adsorbed on the ferromagnetic nanoparticle on which the antibody is immobilized. After adsorption, a labeled antibody (secondary antibody) (secondary antibody) is added, separated from the sample by a magnet, the separated ferromagnetic nanoparticles are washed with a buffer solution, etc., and the excess labeled antibody is removed, Then, the obtained ferromagnetic nanoparticles can be diluted again with a buffer solution or the like, and the antigen can be quantified using a spectroscopic technique or the like in the labeling portion.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, the scope of the present invention is not limited to these Examples.
Table 2 shows a list of measuring instruments used in the following examples.

  UV-Vis is an ultraviolet-visible spectrophotometer, XRD is a powder X-ray diffractometer, SQUID is a superconducting quantum interference element magnetometer, TEM is a transmission electron microscope, and these abbreviations are used hereinafter. write.

Example 1 Synthesis of biotinylated FePt nanoparticles Synthesis of organic ligands Two ligands, biotin-PEG-SH and dopamine-mPEG, have been reported as organic ligands for surface modification of FePt nanoparticles (Rui Hong, Nicholas O. Fischer, Todd Emrick, Vincent M. Rotello, chem. Mater, 2005, 17, 4617; Bryan Parrish and Todd Emrick, Macromplecules, 2004, 37, 5863; Karl Kaiser, Markus Marek, Thomas Haselgrubler, Hansgeorg Schindler, and Hermann J. Gruber, Bioconjugate chem. 1997, 8 , 54). The overall scheme is as follows.

Overall synthesis reaction formula of dopamine-mPEG

Biotin-PEG-SH total synthesis reaction formula

Details of each synthesis step are described below.
(1) Synthesis of poly (ethylene glycol) -succinate

  Poly (ethylene glycol) (2.759 g, 5 mmol), succinic anhydride (1.2525 g, 12.5 mmol), 4- (dimethylamino) pyridine (0.0648 g, 0.5 mmol) were dissolved in 100 ml of chloroform. And heated to reflux for 12 hours. Thereafter, the solvent was distilled off under reduced pressure, and the residue was dissolved in water, washed with a solution mixed in a mixing ratio of hexane: ethyl acetate = 1: 1, and extracted with dichloromethane. The dichloromethane extract was dehydrated and dried with magnesium sulfate, and the magnesium sulfate was filtered off. Then, the dichloromethane was distilled off under reduced pressure, and vacuum drying was performed overnight to obtain poly (ethylene glycol) -succinate (3.3718 g, 5 mmol, 100 %).

(2) Synthesis of dopamine-poly (ethylene glycol)

  In 10 ml of N, N-dimethylformamide, poly (ethylene glycol) -succinic acid ester (1.3202 g, 2 mmol), 1-hydroxybenzotriazole (0.2702 g, 2 mmol), N, N-diisopropylethylamine (0.52 g) 4 mmol) at 0 ° C. and stirred. Thereafter, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (3.902 g, 20 mmol) and dopamine hydrochloride (0.4025 g, 2.1 mmol) were added over 5 minutes. The solution was purged with nitrogen and stirred at 4 ° C. overnight, and N, N-dimethylformamide was distilled off under reduced pressure and dissolved in 150 ml of chloroform. After washing with 1M cooled hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution, water and brine, dehydrated and dried over magnesium sulfate, and filtered off magnesium sulfate. Chloroform was distilled off under reduced pressure, vacuum dried and dopamine-poly (ethylene glycol). ) Was obtained (1.28 g, 1.6 mmol, 80%).

(3) Biotin-NHS synthesis

  D-biotin (0.5292 g, 2.17 mmol), N-hydroxysuccinimide (0.3259 g, 2.83 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride in N, N-dimethylformamide The salt (0.53 g, 2.76 mmol) was purged with nitrogen and stirred overnight. N, N-dimethylformamide was distilled off under reduced pressure, filtered and washed with excess water and methanol to obtain white crystals of biotin-NHS (0.4858 g, 1.423 mmol, 65.7%).

(4) Synthesis of CH ₃ COOH · NH ₂ -PEG ₈₀₀ -NH-Boc

Di-tert-butyl in which O, O′-bis- (2-aminopropyl) -polypropylene glycol 800 (50.7 g, 63.4 mmol) was dissolved in 50 ml of methanol and dissolved in 50 ml of methanol over 30 minutes. pyrocarbonate (Boc ₂ ) (12.04 g, 55.2 mmol) was added dropwise, and the mixture was purged with nitrogen and stirred overnight. Thereafter, 3 ml of acetic acid and 100 ml of toluene were added and distilled off under reduced pressure until 50 ml or less, and 100 ml of toluene was added and distilled off under reduced pressure until 50 ml or less. Next, half of the reaction product was developed by column chromatography (silica gel, developing solvent chloroform / methanol / acetic acid = 90: 10: 0.1) and TLC (developing solvent chloroform / methanol / acetic acid = 100: 30: 2). A portion having an Rf value of 0.29 to 0.4 was taken out, and the above-described vacuum distillation operation using toluene was repeated and stored frozen. Then, 300 ml of water was added thereto, sodium chloride was added in excess, stirred and filtered, and undissolved sodium chloride was removed. Thereafter, extraction was performed 3 times with 200 ml of chloroform, followed by dehydration drying with sodium sulfate all day and night. After filtering off the sodium sulfate, the chloroform was distilled off under reduced pressure to obtain the object by vacuum drying _{CH 3 COOH · NH 2 -PEG 800} -NH-Boc.

(5) Synthesis of biotin-NH-PEG ₈₀₀ -NH-Boc

Previously synthesized _{CH 3 COOH · NH 2 -PEG 800} -NH-Boc (1.39g) and Biotin -NHS (0.59g, 1.73mmol) in a DMF12ml of dehydrated triethylamine _(Et 3 N) 0.3ml In addition, it was purged with argon and stirred overnight at room temperature. 12 ml of water was added thereto and stirred for 2 hours, water was distilled off under reduced pressure, and 15 ml of 200 mM aqueous sodium carbonate solution was added and filtered. Thereafter, sodium chloride was added until saturation, extracted three times with 75 ml of dichloromethane, and dehydrated and dried with sodium sulfate all day and night. After sodium sulfate was filtered off, dichloromethane was distilled off under reduced pressure and vacuum-dried to obtain the target biotin-NH-PEG ₈₀₀ -NH-Boc.

(6) Synthesis of biotin-NH-PEG ₈₀₀ -NH ₂ .HCl

The yellow solution turned orange by dissolving the previously synthesized biotin-NH-PEG ₈₀₀ -NH-Boc in 12 ml formic acid and 0.3 ml water, purging with nitrogen, and stirring overnight. Thereafter, the operation of adding 30 ml of toluene and distilling off under reduced pressure was repeated twice to dissolve in 7 ml of water. Next, ion exchange chromatography is performed under a nitrogen atmosphere (Sephadex C-25, filled with water and then developed with a 50 mM sodium chloride aqueous solution bubbled with nitrogen), and the portion having the amino group reacted by the ninhydrin reaction is taken out and subjected to vacuum distillation. Lastly, it was dissolved in brine, extracted three times with 80 ml of chloroform, and dehydrated and dried overnight with sodium sulfate. After sodium sulfate was filtered off, chloroform was distilled off under reduced pressure, and the target biotin-NH-PEG ₈₀₀ -NH ₂ .HCl (1.37 g, 1.3 mmol) was obtained by vacuum drying.

(7) Synthesis of biotin-NH-PEG ₈₀₀ -SH

Biotin-NH-PEG ₈₀₀ -NH ₂ .HCl (930 mg, 0.78 mmol), 3,3′-dithio (succinimidylpropionate) (800 mg, 2 mmol), 500 μl of triethylamine in 100 ml of tetrahydrofuran under nitrogen atmosphere Dissolved and stirred overnight. Thereafter, the solvent was distilled off under reduced pressure, and 50 ml of buffer A (100 mM sodium chloride, 50 mM sodium dihydrogen phosphate, 1 mM ethylenediaminetetraacetic acid was adjusted to pH 7.5 with sodium hydroxide) was added, followed by filtration. The filtrate was bubbled with nitrogen for 2 minutes, 1,4-dithiothreitol (620 mg, 4 mmol) was added and bubbled for an additional 15 minutes. Thereafter, 0.5 M acetic acid was added to adjust the pH to 4.5, and the solvent was distilled off under reduced pressure to 50 ml. Then, 25 ml of the solution was taken out and subjected to column chromatography under a nitrogen atmosphere (Sephadex G-25, developing solvent buffer B (100 mM sodium chloride, 20 mM sodium acetate adjusted to pH 4.5 with hydrochloric acid), and the red avidin-HABA solution was yellow. Then, the biotin-containing portion that was discolored was taken out, extracted three times with 150 ml of chloroform, dehydrated and dried overnight with sodium sulfate, filtered off sodium sulfate, and then the chloroform was distilled off under reduced pressure to obtain the desired biotin-NH. -PEG ₈₀₀ -SH was obtained.

2. Synthesis of FePt Nanoparticles In this example, FePt nanoparticles were synthesized by employing a liquid phase direct synthesis method using a polyol process. Approach using the polyol as a reducing agent and a solvent, lowering the reaction rate Saishi the reduction of Fe and Pt by using tetraethylene glycol undergoes reductive and ordering at the same time in the liquid layer, directly synthesizing the L1 ₀ ordered phase It is. Moreover, the acetylacetonate (acac) complex was used for the organometallic complex as a precursor to a FePt alloy. It is thought that the reaction is actually carried out in the following process (K. Sato, B. Jeyadevan, K. Tohji, J. Magn. Magn. Mater. 2005 , 289, 1) (Fig. 3 ).

The experimental method is specifically described below.
Fe (acac) ₃ (0.106 g, 0.3 mmol) and Pt (acac) ₂ (0.118 mg, 0.3 mmol) were dissolved in 80 ml of tetraethylene glycol and replaced with nitrogen or argon. Heated to 300 ° C. and heated to reflux for 3.5 hours. After the reaction, the reaction solution was air-cooled to room temperature, a small amount of oleylamine and octanoic acid were added to the reaction solution, a large amount of hexane was added, purged with nitrogen or argon, and heated and refluxed at about 100 ° C. in an oil bath all day and night. Thereafter, the hexane layer was separated, hexane was distilled off under reduced pressure, about 100 ml of ethanol was added to the residue, and the mixture was stored in the refrigerator overnight. The cooled ethanol solution is centrifuged (1500 rpm, 12 minutes), the supernatant is removed, and ethanol is added in the same manner. Centrifugation is performed in the same manner, the supernatant is removed, the black particles remaining at the bottom are vacuum-dried, and the desired oleylamine and FePt nanoparticles with octanoic acid immobilized thereon were obtained.

3. Synthesis of Biotinylated FePt Nanoparticles by Ligand Exchange Reaction The synthesis process of biotinylated FePt nanoparticles by ligand exchange reaction is shown in FIG. Biotinylated FePt nanoparticles were synthesized by a ligand exchange reaction as follows.
FePt nanoparticles immobilized with oleylamine and octanoic acid (38 mg), dopamine-mPEG (32 mg), biotin-PEG-SH (57 mg) were dissolved in 8 ml of dichloromethane, and the mixture was heated and stirred for 2 days in a nitrogen or argon atmosphere ( 30-40 ° C). After the reaction, dichloromethane was once distilled off under reduced pressure, and a small amount of dichloromethane and diethyl ether were added and centrifuged (1500 rpm, 12 minutes). The supernatant was discarded, a small amount of dichloromethane and diethyl ether were added again, and then centrifuged again (1500 rpm, 12 minutes). The supernatant was discarded, and the desired black biotinylated FePt nanoparticles were obtained in the precipitate.

(Example 2) Evaluation of FePt nanoparticles FePt nanoparticles were evaluated through magnetization measurement by SQUID, X-ray diffraction pattern measurement by XRD, and observation of particle size distribution by TEM.

(1) Magnetization measurement by SQUID When measuring the magnetization of FePt nanoparticles, as shown in FIG. 5, a measurement sample was prepared by wrapping FePt nanoparticles with cellophane tape and sandwiching the upper and lower sides with a straw. At this time, since the magnetization of the FePt nanoparticles was very large, it was judged that the diamagnetism due to the straw was almost negligible.
The magnetization curve was measured. That is, the magnetic field dependence of magnetization was measured.
Furthermore, the magnetization measurement by ZFC-FC was performed.

The result of the magnetization measurement at this time is shown in FIG.
As can be seen from this result, a clear hysteresis curve is observed even at 300K, and the presence of remanent magnetization and coercive force confirms the formation of room temperature ferromagnetic nanoparticles. The actual values are as shown in Table 3.

FIG. 7 shows the measurement results of the ZFC-FC magnetization curve.
This result confirms the formation of room temperature ferromagnetic nanoparticles even at 400K.

(2) Measurement synthesized FePt nanoparticles X-ray diffraction pattern by XRD determine whether taking an L1 ₀ ordered phase is very effective to examine the X-ray diffraction pattern by XRD. At this time, if FePt nanoparticles are L1 ₀ ordered phase should disappear naturally by extinction rule should be observed peaks of the grid that are not confirmed in fcc structure. That is, if a lattice reflection peak from the (001), (110), (210), (112) plane, which is not originally observed in the fcc structure, is observed, the FePt nanoparticle has an L1 ₀ ordered phase. Can do. The measurement result by XRD of the FePt nanoparticles actually measured here is shown in FIG.

From the results, (001), (110), (210), since the superlattice peaks not seen originally observed in the fcc structure of (112), FePt nanoparticles created by taking an L1 ₀ ordered phase I understand that.
Moreover, when the lattice constant was calculated using the following formula, the a-axis was 3.85 mm.

  At this time, since the c-axis has Fe and Pt alternately stacked in layers, the value is slightly smaller than that of the a-axis. Met.

  Furthermore, when the average particle diameter was calculated by the Scherrer equation shown below, the average particle diameter was 6.36 nm, and the formation of nanoparticles was confirmed by measurement by XRD.

θ：回折角（°），λ：Ｘ線波長（１．５４Å），ｈ，ｋ，ｌ：ミュラー指数，
ｄ：面間隔（Å），ｋ：固有係数（約０．９），β：半幅値（ｒａｄ）

θ: diffraction angle (°), λ: X-ray wavelength (1.54 mm), h, k, l: Mueller index,
d: surface interval (Å), k: eigencoefficient (approximately 0.9), β: half-width value (rad)

(3) Observation of FePt nanoparticles by TEM In order to confirm the particle size distribution and dispersion of the nanoparticles, observation was performed by TEM.
In the observation method, a sample was prepared by adding and dispersing hexane to FePt nanoparticles on which oleylamine and octanoic acid were immobilized on a grid, and dropping them. Moreover, about the FePt nanoparticle which fix | immobilized biotin by the ligand exchange method, acetone was added and disperse | distributed and the sample was created by dripping similarly. The observation results are as shown in FIG. 9 below.

From these TEM images, it was confirmed that the FePt nanoparticles were dispersed with a uniform interparticle distance, and since the particles were not in an aggregated state, the organic ligand played a role as a spacer between the particles. That is confirmed. Thus, the immobilization of organic ligands on the FePt nanoparticles is confirmed here.
Moreover, what measured the particle size from each TEM image, and calculated | required distribution and an average particle diameter is shown in FIG.
From this, the synthesis of FePt nanoparticles was confirmed, and it became clear that this was not significantly different from the calculated value of Scherrer's equation by XRD.

(Example 3) Evaluation of ligand exchange by IR spectrum It was verified by IR spectrum whether the ligand exchange reaction proceeded.
Here, FePt nanoparticles with oleylamine and octanoic acid immobilized thereon are measured by a tablet method using KBr, biotinylated FePt nanoparticles are measured by casting on a CaF ₂ base, biotin ligand is dissolved in chloroform, CaF Measurements in a liquid cell according to ₂ were made.

(1) Comparison of IR spectra of biotin ligand and biotinylated FePt nanoparticles FIG. 11 shows IR spectra of biotin ligand and biotinylated FePt nanoparticles measured by the method described above.

Here, since the biotin ligand was dissolved in chloroform, the absorption of chloroform appeared at 3000, 2400 cm ^{−1 and the} like. Therefore, in order to compare SH stretching vibration, which is the optimum absorption for confirming whether or not ligand exchange is performed, an enlarged view of absorption at 2500 to 2700 cm ⁻¹ is shown in FIG.

From this, since the SH stretching vibration of 2585 cm ⁻¹ seen in the biotin ligand is not seen in the biotinylated FePt nanoparticles, the immobilization of the biotin ligand on the FePt nanoparticles was confirmed, Further, since it is explained that the Pt-S bond is generated by the following mechanism, this suggests the generation of the Pt-S bond.
R-SH + Pt → RS-S-Pt + 1 / 2H ₂

(2) Comparison of IR spectra of FePt nanoparticles before and after ligand exchange reaction In the case of (1), the FePt nanoparticles were confirmed to disappear from the SH stretching vibration of the ligand. In order to confirm the presence of biotin ligand by comparing the difference in IR spectra before and after the ligand exchange reaction, IR spectra of FePt nanoparticles and biotinylated FePt nanoparticles with immobilized acid were measured (FIG. 13).

In FIG. 13, a 1,950 cm ⁻¹ 1,2,4-trisubstituted benzene ring overtone derived from a biotin ligand and a 1350 cm ⁻¹ glycol deformation angle not found in FePt nanoparticles immobilized with oleylamine and octanoic acid Since vibration was observed in the FePt nanoparticles after the ligand exchange reaction, it was confirmed that a biotin ligand was present on the FePt nanoparticles after the ligand exchange reaction.
This suggested that the biotin ligand was bound to the FePt nanoparticles after the ligand exchange reaction.

(Example 4) Evaluation of biotinylated FePt nanoparticles by zeta potential In order to investigate whether hydrophilicity was successfully imparted by immobilizing biotin ligands on FePt nanoparticles, the charge state of the fine particle surface by zeta potential I investigated.

  Here, if a biotin ligand is immobilized on the FePt nanoparticle, it should be negatively charged by the PEG chain present in the ligand, thereby imparting hydrophilicity to the FePt nanoparticle. (Fig. 14).

  As a result, it was possible to measure that the surface zeta potential of the FePt nanoparticles was -13.74 mV, indicating that the hydrophilicity based on the PEG chain of the FePt nanoparticles was successfully provided.

(Example 5) Evaluation of biotinylated FePt nanoparticles by avidin-HABA method Quantification of biotinylated FePt nanoparticles in solution Zeta potential measurement confirmed that hydrophilicity of biotinylated FePt nanoparticles was imparted and that it was stably dispersed in water. It was confirmed by avidin-HABA method whether or not biotin bound to the avidin was quantitatively bound.
The biotin on biotinylated FePt nanoparticles dispersed in water was quantified by the following method.
The samples measured here are a solution in which biotinylated FePt nanoparticles are dispersed and a biotin solution as a control.

(1) Determination of biotinylated FePt nanoparticles by avidin-HABA method 0.1M sodium dihydrogen phosphate and 0.1M disodium hydrogen phosphate were prepared, and the two were mixed to obtain pH 7.0. A buffer was made. Thereafter, this was used as a solvent.
(i) Preparation of avidin-HABA solution 1.8 mg of HABA dye (4-hydroxyazobenzene-2′-carboxylic acid) was prepared in a 50 ml volumetric flask, and 10 mg of avidin was added thereto to obtain an avidin-HABA solution. .
(ii) Preparation of biotin solution D-biotin (122 mg, 0.5 mmol) was prepared in a 200 ml volumetric flask, 4 ml was taken out of it and prepared in a 50 ml volumetric flask to obtain a biotin solution.
(iii) Quantification of biotin and biotinylated FePt nanoparticles 2 ml of the prepared avidin-HABA solution was placed in a cell, measured by UV-vis spectrum, 10 μl each of biotin and biotinylated FePt solution were added, and the change in the spectrum was observed. did.
The results are shown in FIG.

Next, in order to investigate the degree of decrease in absorption due to the addition of biotin at 500 nm, a calibration curve of absorption at 500 nm is shown in FIG.
This confirms that the biotinylated FePt nanoparticles have a quantitative decrease in absorption at 500 nm, similar to biotin, so that the biotinylated FePt nanoparticle solution can be quantified by the avidin-HABA method, and avidin It became clear that it can be combined quantitatively.

  This method can be quickly and simply used as a method for confirming whether biotin is present, and can be used as a material for determining whether magnetic separation in a solution of FePt nanoparticles described later is possible.

2. Magnetic separation of biotinylated FePt nanoparticles in solution The biotin on the surface of the biotinylated FePt nanoparticle can be quantitatively bound to avidin without any problem, and the presence of biotin on the biotinylated FePt nanoparticle can be confirmed by using the avidin-HABA method. Thus, it was examined whether or not magnetic separation of biotinylated FePt nanoparticles dispersed in a solution was possible. The outline of the experiment is as shown in FIG.

By this method, the magnetically separated FePt nanoparticles are treated as described in 1. above. In the same manner as above, the solution was dropped into a cell of 2 ml of an avidin-HABA solution, and the change in absorption was examined by UV-vis spectrum. Here, if the absorption at 500 nm is decreased by titration, the presence of biotin, that is, the presence of biotinylated FePt nanoparticles becomes clear, and it becomes clear that biotinylated FePt nanoparticles can be magnetically separated.
The results are shown in FIG.

  This result confirms that the absorption of the 500 nm avidin-HABA complex decreases after titration. This reveals that biotin is contained in the magnetically recovered material, so that biotinylated FePt nanoparticles are confirmed to be recovered. Therefore, it became clear from this result that biotinylated FePt nanoparticles can be magnetically recovered in the solution.

It is a figure which shows the relationship between the structural transition and magnetic characteristic in a FePt alloy. It is a state density figure of a FePt alloy. It is a figure which shows the liquid phase direct synthesis method of the FePt nanoparticle by a polyol process. It is a figure which shows the synthetic | combination process of the biotinylated FePt nanoparticle by a ligand exchange reaction. It is a figure which shows the state of the measurement sample in the magnetization measurement of FePt nanoparticle. It is a figure which shows the result of the magnetization measurement by an external magnetic field change. The upper figure is an overall view, and the lower figure is an enlarged view of -10000 to 10000G. It is a figure which shows the measurement result of a ZFC-FC magnetization curve. It is a figure which shows the measurement result by XRD of FePt nanoparticle. It is a figure which shows the observation result by TEM of the FePt nanoparticle by different magnification. The top is for FePt nanoparticles with oleylamine and octanoic acid immobilized thereon, and the bottom is for biotinylated FePt nanoparticles. It is a figure which shows the particle size distribution of FePt nanoparticle. The left is for FePt nanoparticles with oleylamine and octanoic acid immobilized, and the right is for biotinylated FePt nanoparticles. It is a figure which shows the IR spectrum of a biotin ligand and a biotinylated FePt nanoparticle. It is an enlarged view of IR spectrum of a biotin ligand and biotinylated FePt nanoparticles. It is a figure which shows IR spectrum of the FePt nanoparticle which fix | immobilized oleylamine and octanoic acid, and a biotinylated FePt nanoparticle. It is a figure which shows the structure of a biotinylated FePt nanoparticle. It is a figure which shows the fixed_quantity | quantitative_assay result of biotinylated FePt nanoparticle and biotin. It is a figure which shows the analytical curve of the absorption in 500 nm of biotinylated FePt nanoparticle and biotin. It is a figure which shows the outline | summary of the magnetic separation method from the solution of a biotinylated FePt nanoparticle. It is a figure which shows the change of the spectrum by the dripping of the biotinylated FePt nanoparticle separated magnetically.

Claims (8)

  Organic molecule-immobilized ferromagnetic nanoparticles obtained by immobilizing organic molecules on ferromagnetic FePt nanoparticles. The organic molecule-immobilized ferromagnetic nanoparticles according to claim 1, wherein the ferromagnetic FePt nanoparticles have an L1 ₀ ordered structure.   3. The organic molecule-immobilized ferromagnetic nanoparticles according to claim 1, wherein the ferromagnetic FePt nanoparticles are obtained by treating an Fe complex and a Pt complex with a polyol solvent having a boiling point of 300 ° C. or higher.   The organic molecule-immobilized ferromagnetic nanoparticles according to any one of claims 1 to 3, wherein the organic molecule immobilized on the ferromagnetic FePt nanoparticles is selected from a specific binding partner and a physiologically active substance.   After processing the ferromagnetic FePt nanoparticles with fatty acid and aliphatic amine and immobilizing the fatty acid and aliphatic amine on the ferromagnetic FePt nanoparticles, processing with an organic compound having a catechol residue and an organic compound having a mercapto group The manufacturing method of the organic molecule fixed ferromagnetic nanoparticle of any one of Claims 1-4 characterized by performing.   6. The production method according to claim 5, wherein at least one of the organic compound having a catechol residue and the organic compound having a mercapto group has a residue selected from a specific binding partner, DNA, an enzyme, and a physiologically active substance.   Organic molecule-immobilized ferromagnetic nanoparticles obtained by the production method according to claim 5 or 6.   The organic molecule-immobilized ferromagnetic nanoparticles according to any one of claims 1 to 4 and 7, and / or magnetism from a solution containing particles in which another substance is bound to the organic molecule-immobilized ferromagnetic nanoparticles. Separating the organic molecule-immobilized ferromagnetic nanoparticles and / or particles obtained by binding other substances to the organic molecule-immobilized ferromagnetic nanoparticles by the method described above.

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