Editing Magnetic Nanoparticles

aminobutyl-NTA<br>

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Styrene(St) was washed two times by 10% NaOH solution, dried<br>by calcium chloride and distilled under reduced pressure before<br>use. Azo-bis-isobutryonitrile (AIBN) was recrystallized twice<br>from absolute ethanol. Maleic anhydride(MAn) was used as<br>�<br>received. Methacrylic acid was dried over 4 A molecular sieves<br>and distilled under reduced pressure.Toluene was dried over<br>calcium chloride. p-Divinyl benzene (DVB) and all other<br>reagents were used as received without further purification.<br>Preparation of Fe3O4 nanoparticles<br>The Fe3O4 spheres were prepared by the solvothermal reaction.<br>270 mg of FeCl3 $ 6H2O, and 328 mg of NaOAc were dissolved in<br>8 mL of ethylene glycol, followed by the addition of 8 mL of poly<br>ethylene glycol (600) under stirring. The obtained homogeneous<br>yellow solution was sealed in Teflon-lined stainless-steel auto-<br>claves, heated from 30 � C to 180 � C for 2 h, and maintained at<br>180 � C for 12 h. The obtained Fe3O4 spheres were washed by<br>ethanol and water for several times, and finally re-dispersed in<br>ethanol (8 mL) for further use.<br>Preparation of Fe3O4@SiO2 core-shell particles<br>The Fe3O4@SiO2 Core-shell spheres were prepared according to<br>the St€ber method. 40 mg of Fe3O4, 6 mL of concentrated<br>o<br>ammonia and 80 mL of water were mixed with 320 mL of<br>ethanol. After ultrasonication for 10 min, 0.22 mL of tetrae-<br>thoxysilane(TEOS) was added to the mixture with continuous<br>stirring. After the reaction for more than 8 h, the obtained<br>product was collected by a magnet, washed by water and abso-<br>lute ethanol, and re-dispersed in 4 mL of ethanol for further use.<br>Preparation of Fe3O4@SiO2-NH2 core-shell particles<br>40 mg of Fe3O4@SiO2 core-shell particles, 40 mL of ethanol, and<br>0.04 mL of 3-aminopropyltrimethoxysilane (APS) were mixed<br>and refluxed overnight. The Fe3O4@SiO2-NH2 particles were<br>separated with a magnet and washed with ethanol and DMF,<br>and finally re-dispersed in 4 mL of DMF for subsequent use.<br>Preparation of Fe3O4@SiO2/NTA particles<br>2.0 mg of Fe3O4@SiO2-NH2 particles were dispersed in the PBS<br>solution (1.5 ml) with ultrasonication. Thereafter, 25% glutaral-<br>dehyde (GLH) solution was added and the reaction was aged for<br>8 h at room temperature. The magnetic particles were washed with<br>PBS for three times and dispersed in the PBS solution (1.0 mL),<br>followed by the addition of 1.2 mg of nitrilotriacetic acid (NTA).<br>After more than 8 h, 0.5 mg of NaBH4 was added to the above<br>solution. After stirred for 2 h, the products were separated, washed<br>with PBS, and re-dispersed in PBS for subsequent use.<br>Preparation of Fe3O4@SiO2/P(St-alt-MAA)/NTA particles<br>5.0 mg of Fe3O4@ SiO2-NH2 particles, 50 mL of methacrylic acid<br>and 0.1 g of dicyclohexylcarbodiimide (DCC) were mixed in<br>5.0 mL of dry DMF. After stirred for 8 h at 40 � C, the particles<br><br> 

were separated, washed with DMF and dispersed in 10 mL of<br>toluene. The obtained dispersion was then transferred into<br>a three-necked flask together with MAn (0.15g, 1.5 mmol), St<br>(0.16 mL, 1.4 mmol) and p-divinyl benzene (DVB) (7.5 mL). After<br>degassing with nitrogen for 10 min, the solution was heated up to<br>75 � C. 0.5 mL AIBN solution (20 mg/mL) was injected to the<br>mixture. After reaction for 1.5 h, the magnetite/polymer core/<br>shell structure were obtained, and washed with dry DMF for five<br>times to remove the physioabsorbed polymer, and then redis-<br>persed in 1.0 mL of PBS solution containing of NTA (1.2 mg)<br>and triethylamine (5.0 mL). After 8 h of reaction, the final<br>particles were separated, washed with PBS, and re-dispersed in<br>PBS for subsequent use.<br>Preparation of Fe3O4@SiO2/Ni-NTA and Fe3O4@SiO2/P<br>(St-alt-MAA)/Ni-NTA Particle<br>Fe3O4@SiO2/NTA or Fe3O4@SiO2@poly(styrene-alt-maleic<br>acid)/NTA (Fe3O4@ SiO2/P(St-alt-MAA)/NTA) solution was<br>added 1.0 mL Ni(CH3COO)2 solution (0.1 M) and shaken for<br>1 h. Then, the product was separated from the solution and<br>washed two times with PBS.<br>Activity of the nanoparticles for separation of His-tagged<br>proteins<br>20 mg of Fe3O4@SiO2/Ni-NTA or Fe3O4@SiO2/P(St-alt-MAA)/<br>Ni-NTA particles were added to 6.0mg of His-tagged GFP or<br>OFP without his-tag solution (PBS buffer, 10mL) and incubated<br>for 10 min on the ice. The particles were separated by a magnet,<br>and incubated with imidazole solution (3 M, 25mL) for 10 min to<br>release the captured proteins.<br>Recyclability of magnetic spheres for separation of His-tagged<br>proteins<br>Magnetic spheres used were collected for recycling experiment.<br>In a typical procedure, 20 mg of Fe3O4@SiO2/Ni-NTA or 12 mg<br>of Fe3O4@SiO2/P(St-alt-MAA)/Ni-NTA particles were washed<br>with 10 ml PBS two times, then incubated with 50 mM EDTA<br>in PBS buffer for 20 min, mixed with Ni(CH3COO)2 solution<br>(0.1 M, 20 mL). After washed with 10 mL PBS, the magnetic<br>spheres were incubated with His-tagged GFP solution to inves-<br>tigate their separation performance.<br><br> 

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The Fe(acac)3, oleic acid, oleyl amine and 1,2-hexadecanediol were obtained from Aldrich,<br>Pt(acac)2 from Strem Chemicals, dioctyl ether from SAFC, ethanol from AAPER and hexane from<br>EMD, which were used without further purification. Synthesis was carried out under standard<br>airless techniques in an argon atmosphere. In a typical synthesis route, Pt(acac)2 and Fe(acac)3 in<br>the molar ratio 1:1 were taken in a 125 mL European flask containing a PTFE coated magnetic stir<br>bar at room temperature. 1, 2-hexadecanediol (5 times mole amount of Pt(acac)2 and Fe(acac)3) was<br>added to the flask. 30 mL of dioctyl ether was then transferred into the flask and the contents were<br>stirred while purging with Ar for 30 minutes at room temperature. The flask was then heated to 200o<br>C at 6o C per minute by use of a Glas-Col hemispherical heating mantel connected to a<br>programmable heat controller using a type J thermocouple. Once the temperature reached 200o C,<br>the flask was kept at this temperature for 30 minutes. After the 30 minute hold, the flask was<br>heated to 295o C at a rate of approximately 5o C per minute. The flask was maintained at a refluxing<br>temperature of 295o C for 30 minutes before cooling down to room temperature under the Ar purge.<br>Afterwards, all handling was performed open to the atmosphere. For comparison purpose FePt of<br>average 2 nm size having Pt(acac)2 to Fe(acac)3 molar ratio of 1, was prepared similar to the method<br>as reported earlier [18].<br>Purification of the nanoparticles was accomplished as follows: 5 mL of the dispersion taken from<br>the flask was added to 20-25 mL of ethyl alcohol (EtOH) and the mixture was centrifuged (6000<br>rpm for 15 minutes). The supernatant was discarded and the precipitate was redispersed in 10 mL of<br>hexane. The dark brown dispersion was stored under refrigeration at approximately 10o C.<br>Samples for magnetic characterization were prepared by depositing a few drops of the hexane<br>dispersion of the sample onto a silicon substrate and allowing the solvent to evaporate at room<br>temperature. In order to transfer the disordered face-centered cubic (fcc) structure of the as-<br>synthesized FePt nanoparticles to L10 structure with high magnetocrystaline anisotropy, the<br>deposited thin film samples were annealed either in a nitrogen or forming gas (Ar + 7% H2)<br>atmosphere for 1 h at temperatures between 550 and 900o C. Magnetic hysteresis loops of the<br>samples were measured by a superconducting quantum interference device (SQUID). Powder X-<br>ray diffraction (XRD) patterns were measured using a Phillips MPD diffractometer with a CuKα<br>source (λ = 1.5405 Å) and transmission electron microscopy (TEM) measured by a JEOL 1200 EX<br>electron microscope at an accelerating voltage of 120 kV.<br><br>One typical synthetic procedure is as follows: under airless condition, platinum acetylacetonate (197 mg, 0.5 mmol), 1,2-hexadecanediol (390 mg, 1.5 mmol), and dioctylether (20 ml) were mixed and heated to 100°C. Oleic acid (0.16 ml, 0.5 mmol), oleyl amine (0.17 ml, 0.5 mmol), and Fe(CO) 5 (0.13 ml, 1 mmol) were added, and the mixture was heated to reflux (297°C). The refluxing was continued for 30 min. The heat source was then removed, and the reaction mixture was allowed to cool to room temperature. The inert gas protected system could be opened to ambient environment at this point. The black product was precipitated by adding ethanol (∼40 ml) and separated by centrifugation. Yellow-brown supernatant was discarded. The black precipitate was dispersed in hexane (∼25 ml) in the presence of oleic acid (∼0.05 ml) and oleyl amine (∼0.05 ml) and precipitated out by adding ethanol (∼20 ml) and centrifuging. The product was dispersed in hexane (∼20 ml), centrifuged to remove any unsolved precipitation (almost no precipitation was found at this stage), and precipitated out by adding ethanol (∼15 ml) and centrifuging. The materials were redispersed in hexane and stored under N2. The total weight of material recovered was 162 mg. <br> 

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0.02 ml of hexane-dispersed FePt nanoparticles (equivalent to ∼5 mg/ml) was deposited on a SiO2/Si substrate (∼0.5 cm by 1 cm). The solvent was allowed to evaporate slowly (∼5 min) at room temperature. The as-deposited thin film was then transferred into a N2 glove box (O2 concentration &lt;2 ppm) and annealed in a Thermolyne 1300 furnace. The temperature was raised to 550°C from room temperature over a period of 13 min and maintained for 30 min. The sample was taken out of the furnace and cooled to room temperature in the N2 box. Composition and thickness of the FePt nanocrystal assembly were determined by Rutherford backscattering spectrometry. This procedure yielded a ∼120-nm-thick FePt nanocrystal assembly. <br> 

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One mL of iron oxide magnetic nanoparticles suspension (16.3 mg/mL) was added to ligand 8 (0.5 mL of a<br>10 mg/mL MeOH solution in 1.25 mL of distilled water). The two layers were sonicated for 1<br>hour, and repeatedly washed with n-hexanes, then methanol until the nanoparticles were readily<br>dispersed in distilled water. Using a magnet, the nanoparticles were collected and placed in 2<br>mL of distilled water. The mono-NTA modified nanoparticles (0.5 mL) were placed in 1M<br>NiCl2 solution and sonicated for 1 hour. The divalent Ni(II)-loaded nanoparticles were stored in<br>the NiCl2 solution before use.<br> 

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Iron oxide nanoparticle surface modification with mono-NTA Ni(II) chelate. One mL of<br>iron oxide magnetic nanoparticles suspension (16.3 mg/mL) was added to ligand 8 (0.5 mL of a<br>10 mg/mL MeOH solution in 1.25 mL of distilled water). The two layers were sonicated for 1<br>hour, and repeatedly washed with n-hexanes, then methanol until the nanoparticles were readily<br>dispersed in distilled water. Using a magnet, the nanoparticles were collected and placed in 2<br>mL of distilled water. The mono-NTA modified nanoparticles (0.5 mL) were placed in 1M<br>NiCl2 solution and sonicated for 1 hour. The divalent Ni(II)-loaded nanoparticles were stored in<br>the NiCl2 solution before use.<br><br>The Ni(II)-loaded nanoparticles (10 μL; 16.3 mg/mL) were washed in distilled<br>water (3×50 μL) and wash buffer (3×50 μL; 50 mM phosphate buffer system, 300 mM NaCl, 10<br>mM imidazole, pH 8.0). The washed nanoparticles were placed in 40 uL of wash buffer and 10<br>uL of 6×His-Ub (1 μg 6×His-Ub/1 μL of distilled water) and incubated for 60 minutes at 4 °C.<br>Using a centrifuge and magnet, the supernatant was collected and nanoparticles washed in 50 μL<br>aliquots (FT, W1, W2, W3). After W3, 50 μL of elution buffer (50 mM phosphate buffer<br>system, 300 mM NaCl, 250 mM imidazole, pH 8.0) was added and rotated for 10 minutes at<br>4°C. Nanoparticles were collected in 50 μL aliquots (E1, E2). Non-specifically bound protein<br>was released from the nanoparticles by boiling at 95 °C for 4 minutes. The volumes of the<br>aliquots were reduced for analysis by SDS-PAGE (12.5% separation gel, 180V, 50 min). The<br>SDS-PAGE gel was stained using Coomassie Blue overnight.<br><br> 

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