Method of manufacturing superparamagnetic nanocomposite and superparamagnetic nanocomposite manufactured using the same

Abstract

The present invention relates to a method of manufacturing a superparamagnetic nanocomposite and a superparamagnetic nanocomposite manufactured using the same, and more particularly to a method of manufacturing a superparamagnetic nanocomposite suitable for use in magnetic separation for the detection of a target biomaterial and a superparamagnetic nanocomposite manufactured using the same. The method of manufacturing the superparamagnetic nanocomposite according to the present invention has a higher yield and a high rate without complicated processing than a conventional method of manufacturing a magnetic nanoparticle for magnetic separation and is capable of mass production of the superparamagnetic nanocomposite having excellent properties with uniform size and particle size distribution, high aqueous solution dispersibility and high magnetization and being capable of maintaining superparamagnetism.

Claims

1. A superparamagnetic nanocomposite comprising a magnetic nanocrystal which is Fe.sub.3O.sub.4 having a diameter of from more than 0 to 10 nm, wherein a surface of the magnetic nanocrystal is stabilized by carboxylate (COO—) group of dicarboxyl poly(ethylene glycol) having a molecular weight of 500 to 50,000, wherein the superparamagnetic nanocomposite has a plurality of magnetic nanocrystals clustered therein, has a nanoclustered shape having a diameter of 200 nm to 450 nm and has hydrophilicity so as to be dispersed in an aqueous solution.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 schematically shows a method of manufacturing a superparamagnetic nanocomposite (particularly, superparamagnetic iron oxide nanocomposite) having magnetic nanoclustered shape according to Examples 1 and 2;

(2) FIG. 2 shows the results of SEM observation of the superparamagnetic nanocomposites of Examples 1 and 2;

(3) FIG. 3 shows the results of measurement of the size of the superparamagnetic nanocomposite of Example 1;

(4) FIG. 4 shows the results of measurement of the zeta potential of the superparamagnetic nanocomposite of Example 1;

(5) FIG. 5 shows the results of measurement of the size of the superparamagnetic nanocomposite of Example 2;

(6) FIG. 6 shows the results of measurement of the zeta potential of the superparamagnetic nanocomposite of Example 2;

(7) FIG. 7 shows the results of measurement of the magnetism of the superparamagnetic nanocomposite of Example 1;

(8) FIG. 8 shows the results of measurement of the magnetism of the superparamagnetic nanocomposite of Example 2; and

(9) FIG. 9 shows tube photographs before and after isolation of red blood cells (RBCs) and microscope images of the separated superparamagnetic nanocomposite and the supernatant.

MODE FOR INVENTION

(10) A better understanding of the present invention will be given through the following Examples, which are merely set forth to illustrate, but are not to be construed as limiting the present invention and may be variously altered and modified.

(11) In the present invention, a superparamagnetic nanocomposite refers to superparamagnetic particles having nanoclustered shape having a diameter of 100 to 450 nm, preferably 150 to 400 nm, and more preferably 200 to 350 nm configured such that single magnetic particles having a diameter of several nanometer (a diameter of from more than 0 to 10 nm), that is, magnetic nanocrystals, are clustered therein.

(12) In the present invention, “room temperature” may refer to, but is not limited to, 15 to 25° C., which enables the most easily practicable reaction by a worker because increasing or decreasing the temperature is not necessary. Depending on the surrounding conditions and environments, the same may be a temperature higher or lower than the above range.

(13) Here, superparamagnetism is a property that may be controlled using a magnetic force and enables re-dispersion in the absence of a magnetic force, and a superparamagnetic nanocomposite may be utilized in diverse fields requiring magnetic nanoparticles having superparamagnetism.

Reference Example 1. Material Preparation

(14) Ferric chloride hexahydrate (FeCl.sub.3.6H.sub.2O, ACS reagent, 97%, MW=270.30), trisodium citrate dihydrate (HOC(COONa)(CH.sub.2COONa).sub.2.2H.sub.2O; C.sub.6H.sub.5Na.sub.3O.sub.7, 99%, MW=294.10), sodium acetate anhydrous (C.sub.2H.sub.3NaO.sub.2, MW=82.03), and ethylene glycol anhydrous (C.sub.2H.sub.6O.sub.2, 99.8/%, MW=62.07) were purchased from Sigma-Aldrich (St. Louis, Mo., USA), and dicarboxyl poly(ethylene glycol) (PEG-diacid; polyethylene glycol diacid) (COOH-PEG-COOH, MW=2000) was purchased from Jenkem Technology (Beijing, China). Ultrapure water (Nanopure H.sub.2O; >18.0 MΩ) was purified using a Milli-Q water purification system.

Example 1. Method of Manufacturing Superparamagnetic Nanocomposite

(15) A superparamagnetic nanocomposite (particularly a superparamagnetic iron oxide nanocomposite) having magnetic nanoclustered shape of Example 1 was synthesized using a method shown in FIG. 1.

(16) 1.08 g (0.1 M, 3.996 mmol) of FeCl.sub.3.6H.sub.2O was dissolved in 40 mL of ethylene glycol and stirred for 30 min. Thereafter, 0.8 g (0.034 M, 2.720 mmol) of trisodium citrate dihydrate (TSC) was added to the stirred mixture and then further stirred at a rate of 900 rpm for 1 hr. After confirmation of completion of the complete dissolution of trisodium citrate, 2.4 g (0.731 M, 58.515 mmol) of sodium acetate was added to the mixture, and further stirred at 900 rpm for 30 min. Thereafter, the stirred mixture was placed in a Teflon tube for hydrothermal synthesis, enveloped with a stainless steel container so as to be hermetically scaled, placed in a hydrothermal synthesizer, heated from room temperature to 200° C. at a rate of 7° C./min, and reacted at 200° C. for 8 hr to 12 hr while maintaining the temperature at 200° C. When the temperature reached the maximum temperature (200° C.), the inner pressure of the sealed synthesis tube was maintained at 1.5 to 2.5 bar.

(17) In the product synthesized through hydrothermal synthesis reaction, the supernatant was removed through magnetic separation, and the synthesized particles were washed with 30 mL of ethanol five times and deionized water five times and then dried, thus yielding a superparamagnetic nanocomposite. The magnetic separation was performed in a manner in which a sample was placed on a neodymium permanent magnet and particles were collected to thereby remove the supernatant, thus separating the particles. The synthesized particles may also be separated through centrifugation.

(18) As shown in FIG. 1, magnetic nanocrystals, that is, magnetite nanocrystals configured such that the surface thereof is stabilized by the carboxylate (COO.sup.−) group of the trisodium citrate dihydrate molecule (i.e. by chemisorbing or anchoring the carboxylate (COO.sup.−) group of the trisodium citrate dihydrate molecule and the Fe—OH group) were formed, and the particles were negatively charged by the carboxylate (COO.sup.−) group of the trisodium citrate dihydrate molecule to thus cause electrostatic repulsion, and were thus stabilized. Meanwhile, surface tension simultaneously acted in the clustering direction, thereby decreasing the high surface energy of the magnetic nanocrystals, and a superparamagnetic nanocomposite having a uniform size was formed through the balance of electrostatic repulsion and surface tension.

Example 2. Method of Manufacturing Superparamagnetic Nanocomposite

(19) A superparamagnetic nanocomposite (particularly a superparamagnetic iron oxide nanocomposite) having magnetic nanoclustered shape of Example 1 was synthesized using a method shown in FIG. 1.

(20) 2.16 g (0.2 M, 7.991 mmol) of FeCl.sub.3.6H.sub.2O was dissolved in 40 mL of ethylene glycol and stirred for 30 min. Thereafter, 0.8 g (0.034 M, 2.720 mmol) 0.02 g (0.25 mM, 0.01 mmol) of PEG-diacid (polyethylene glycol diacid having MW of 2000) was added to the stirred mixture and then further stirred for 1 hr. After confirmation of completion of the complete dissolution of PEG-diacid, 2.4 g (0.731 M, 58.515 mmol) of sodium acetate was added to the mixture and further stirred for 30 min. Thereafter, the stirred mixture was placed in a Teflon tube for hydrothermal synthesis, enveloped with a stainless steel container so as to be hermetically sealed, placed in a hydrothermal synthesizer, heated from room temperature to 200° C. at a rate of 7° C./min, and reacted at 200° C. for 8 hr to 12 hr while maintaining the temperature at 200° C. When the temperature reached the maximum temperature (200° C.), the inner pressure of the sealed synthesis tube was maintained at 1.5 to 2.5 bar.

(21) In the product synthesized through hydrothermal synthesis reaction, the supernatant was removed through magnetic separation, and the synthesized particles were washed with 30 mL of ethanol five times and deionized water five times and then dried, thus yielding magnetic nanoparticles. The magnetic separation was performed in a manner in which a sample was placed on a neodymium permanent magnet and particles were collected to thereby remove the supernatant, thus separating the particles. The synthesized particles may also be separated through centrifugation.

(22) As shown in FIG. 1, magnetic nanocrystals, that is, magnetite nanocrystals configured such that the surface thereof is stabilized by the carboxylate (COO.sup.−) group of the PEG-diacid molecule (i.e. by chemisorbing or anchoring the carboxylate (COO.sup.−) group of the PEG-diacid molecule and the Fe—OH group) were formed, and the particles were negatively charged by the carboxylate (COO.sup.−) group of the PEG-diacid molecule to thus cause electrostatic repulsion, and were thus stabilized. Meanwhile, surface tension simultaneously acted in the clustering direction, thereby decreasing the high surface energy of the magnetic nanocrystals, and a superparamagnetic nanocomposite having a uniform size was formed through the balance of electrostatic repulsion and surface tension.

Test Example 1. Physicochemical Properties of Superparamagnetic Nanocomposite

(23) 1-1. Observation of Shape of Superparamagnetic Nanocomposite Using SEM (Scanning Electron Microscope)

(24) The superparamagnetic nanocomposites of Examples 1 and 2 were observed to determine the size and shape thereof using a SEM (S-4700, Hitachi, Tokyo, Japan). The results are shown in FIG. 2.

(25) FIG. 2 shows the results of SEM observation of the superparamagnetic nanocomposites of Examples 1 and 2.

(26) As shown in the SEM images of FIG. 2, the clustered structure of nanocrystals was observed on the surface of the cluster, and the particle size distributions of the superparamagnetic nanocomposites were 305.9±24.7 nm in Example 1 and 241.7±20.1 nm in Example 2.

(27) As is apparent from the SEM images of the superparamagnetic nanocomposites of Examples 1 and 2, relative standard deviation upon measurement of the distribution of the superparamagnetic nanocomposites fell within 15%.

(28) Therefore, the superparamagnetic nanocomposites of Examples 1 and 2 can be confirmed to have uniform size and particle size distribution.

(29) 1-2. Analysis of Size, Distribution and Surface Zeta Potential of Superparamagnetic Nanocomposites

(30) The size, distribution and surface zeta potential of the superparamagnetic nanocomposites of Examples 1 and 2 were measured using a Zetasizer (Nano ZS, available from Malvern) through dynamic light scattering particle size analysis. The superparamagnetic nanocomposites of Examples 1 and 2 were analyzed for average hydrodynamic diameter, PDI (polydispersity index) {PDI=(standard deviation of particle size/average particle size).sup.2} and surface zeta potential (three repeated measurements). The results are shown in Tables 1 and 2 below and in FIGS. 3 to 6.

(31) Specifically, Tables 1 and 2 show the results of average hydrodynamic diameter, PDI and surface zeta potential of the superparamagnetic nanocomposites of Examples 1 and 2, respectively. FIG. 3 shows the results of measurement of the size of the superparamagnetic nanocomposite of Example 1, FIG. 4 shows the results of measurement of the zeta potential of the superparamagnetic nanocomposite of Example 1, FIG. 5 shows the results of measurement of the size of the superparamagnetic nanocomposite of Example 2, and FIG. 6 shows the results of measurement of the zeta potential of the superparamagnetic nanocomposite of Example 2.

(32) TABLE-US-00001 TABLE 1 Classification Size (nm) PDI Zeta potential (mV) 1.sup.st Measurement 255.8 0.067 −15.4 2.sup.nd Measurement 254.3 0.086 −15.0 3.sup.rd Measurement 252.6 0.066 −15.0 Average 254.2 0.073 −15.1

(33) TABLE-US-00002 TABLE 2 Classification Size (nm) PDI Zeta potential (mV) 1.sup.st Measurement 268.7 0.100 +24.3 2.sup.nd Measurement 273.1 0.136 +25.7 3.sup.rd Measurement 273.4 0.077 +26.0 Average 271.7 0.104 +25.3

(34) For the PDI of the superparamagnetic nanocomposites of Examples 1 and 2, based on a nearly monodisperse sample in the PDI range of 0 to 0.1, a mid-range polydisperse in the PDI range of 0.1 to 0.7, and sedimentation in the PDI range of >0.7, respective PDI values of Examples 1 and 2 were 0.073 and 0.104, falling in the range of less than 0.1 to about 0.1, corresponding to nearly monodisperse. Briefly, the superparamagnetic nanocomposites of Examples 1 and 2 had a uniform size and particle size distribution.

(35) Also, respective zeta potentials of the superparamagnetic nanocomposites of Examples 1 and 2 were −15.1 mV and +25.3 mV, falling in the zeta potential range of ±10-30 mV, from which the superparamagnetic nanocomposite particles are evaluated to be efficiently dispersed through electrostatic repulsion. The superparamagnetic nanocomposites of Examples 1 and 2 were stabilized by the carboxylate (COO.sup.−) group, and thus had a zeta potential of ±10-30 mV, thereby exhibiting high dispersibility in an aqueous solution.

(36) 1-3. Analysis of Magnetism of Superparamagnetic Nanocomposites

(37) In order to evaluate whether the superparamagnetic nanocomposites of Examples 1 and 2 have high magnetization with superparamagnetism and thus have high separation capability suitable for use in magnetic separation, magnetism was measured using a superconductive quantum interference device. The results are shown in FIGS. 7 and 8. FIGS. 7 and 8 show the magnetic hysteresis loops of the superparamagnetic nanocomposites, as the result of measurement of magnetism.

(38) As shown in FIGS. 7 and 8, the superparamagnetic nanocomposites of Examples 1 and 2 exhibited superparamagnetism at a temperature of 300 K, and saturation magnetization values of Example 1 and 2 were 76 emu/g and 87 emu/g, respectively.

(39) A ferromagnetic material is unsuitable for use in magnetic nanoparticle-mediated isolation technology because clustering of particles may strongly occur when a ferromagnetic material, having high residual magnetization, is repeatedly subjected to an external magnetic field. However, it is typically difficult to maintain the superparamagnetism of magnetic materials at room temperature. In particular, superpara-ferromagnetism transition occurs readily depending on how efficiently single domains are adjusted, regardless of the structures of magnetic materials or magnetic composites. Although the superparamagnetic nanocomposites of Examples 1 and 2 according to the present invention are configured such that tens of thousands of superparamagnetic nanocrystals are clustered, a final cluster, that is, a superparamagnetic nanocomposite having a diameter of 200 to 300 nm does not cause ferromagnetism transition, but superparamagnetism thereof is efficiently maintained.

(40) Also, the superparamagnetic nanocomposites of Examples 1 and 2 according to the present invention have high magnetization and thus high separation capability suitable for use in magnetic separation.

Test Example 2. Analysis of Magnetic Separation Capability of Superparamagnetic Nanocomposite

(41) 2-1. Isolation Test of Red Blood Cells (RBCs) in Whole Blood Using Superparamagnetic Nanocomposite

(42) In order to evaluate the magnetic separation capability of the superparamagnetic nanocomposite of Example 1, a test was performed in a manner in which RBCs were captured and isolated from whole blood to thus measure the separation capability thereof.

(43) The surface of the superparamagnetic nanocomposite of Example 1, configured such that magnetic nanocrystals were clustered, was functionalized with an anti-RBC antibody (Fitzgerald, Human RBC antibody, Cat #20R-RR006) through an EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide)/sulfo-NHS (N-hydroxysulfosuccinimide)-mediated covalent-bond-inducing process. Specifically, 10 mg/500 μL of superparamagnetic nanocomposite particles were added with 10 mg/mL of EDC and 50 μL of sulfo-NHS, stirred at room temperature for 15 min, added with 1 mg of anti-RBC antibody, stirred at room temperature for 2 hr, and then washed five times with PBS (Phosphate-buffered saline) (pH 7.4). A PBS (pH 7.4) containing 0.5 mg/25 μL of the anti-RBC antibody-functionalized superparamagnetic nanocomposite was added to 25 μL of a whole blood sample, reacted at room temperature for 5 min, and then subjected to magnetic separation, thus separating the anti-RBC antibody-functionalized superparamagnetic nanocomposite. Thereafter, the separated anti-RBC antibody-functionalized superparamagnetic nanocomposite and the supernatant were observed under a microscope to count the number of RBCs, and the RBC separation capability (%) was calculated based on Equation 1 below. The results are shown in FIG. 9.

(44) FIG. 9 shows the tube photographs before and after separation of RBCs and the microscope images of the separated superparamagnetic nanocomposite and the supernatant.
RBC separation capability (%)=number of captured RBCs/(number of captured RBCs+number of noncaptured RBCs)×100  [Equation 1]

(45) As shown in FIG. 9, in the left tube photograph, in which the whole blood sample and the anti-RBC antibody-functionalized superparamagnetic nanocomposite were mixed, RBCs were captured through a specific reaction with the antibody of the surface of the superparamagnetic nanocomposite, and the magnetic particles were isolated by an external magnetic field, whereby whole blood, which is red in color, was separated by the superparamagnetic nanocomposite and became transparent. When the separated RBCs and the superparamagnetic nanocomposite bound to RBCs by the antibody are observed under a microscope, RBCs (pale color) and the superparamagnetic nanocomposite (relatively deep black) are seen to efficiently bind to each other, and no portion where only RBCs were present was observed. Moreover, the microscope image of the supernatant showed that RBCs were not observed.

(46) The RBC separation capability of the superparamagnetic nanocomposite was calculated to be 99.5% based on Equation 1. Thereby, the target RBCs alone were specifically captured and isolated from whole blood containing an excess of non-target immunoglobulin within a short reaction time of 5 min using the magnetic particles, and were thus rapidly separated.