Editing Magnetic beads Preparation Methods (section)

== Preparation of NiFe2O4 Nanoparticles ==

NiFe2O4 NPCs were synthesized by a hydrothermal process using nickel chloride hexahydrate (NiCl2$6H2O, ReagentPlus, Sigma-Aldrich) and iron chloride hexahydrate (FeCl3$6H2O, 97.0%, Samchun Chemicals) as the precursors.17,21,22 Ethylene glycol (99.5%, Samchun Chemicals) and sodium acetate (anhydrous, 99.0%, Sigma-Aldrich) were used for the formation of NPCs in this polyol process. The detailed roles of these reactants have been described in previous reports.21,23,24 First, 0.36 g (1.5 mmol) of NiCl2.6H2O and 0.81 g (3.0 mmol) of FeCl3.6H2O were dissolved in 30 mL of ethylene glycol and stirred until a clear solution was formed. Then, 2.16 g of sodium acetate was added to it with vigorous stirring. After 30 min, a KNO3 solution (0.30 g of KNO3 in 2 mL of distilled water) was added and stirred for 10 min. The obtained solution was transferred to a Teflonlined stainless steel autoclave (100 mL capacity) and sealed. The autoclave was heated at 200 oC for 8 h and then naturally cooled for 30 min followed by forced cooling to room temperature (25 oC) in a flowing water bath. The obtained particles were washed several times with ethanol, and then dried at 60 oC for 10 h. spherical NPCs with diameters of approximately 100 nm. The result of dynamic light scattering analysis shows that the average size of NPCs in aqueous dispersion was ~150 (~45) nm.



Binding: 50 mM&nbsp; Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0&nbsp;<br/>buffer I: 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, and pH 8.0&nbsp;&nbsp;<br/>Wash buffer II: 50 mM Tris-HCl, 300 mM NaCl, 40 mM imidazole, and pH 8.0&nbsp;<br/>Elution buffer: 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, and pH 8.0 for 10 min, recovery: (60%) were recovered<br/>Reclycling: can reuse 7 times<br/>Capacity: 100 mg of Histagged sGFP can bind to 1 g of NiFe2O4 NPCs



Briefly, 0.2 g FeCl3 and 0.073 g FeCl2 were dissolved in 1 mol L−1 HCL (12 mL). Then, 0.05 g agarose was dissolved in 2 mol L−1 NaOH (25 mL) under heating to 80 ◦C. The alkaline agarose solution was deoxygenated by purging nitrogen and 2 mL of the Fe3+/Fe2+ solution was added to it, dropwise. The addition was made under nitrogen atmosphere at reflux conditions (90 ◦C) and magnetic stirring (800 rpm). The color of the bulk solution changed to black immediately, that indicates the production of Fe3O4 nanoparticles. By this method, magnetic micro-flake agarose particles were produced. The final mixture was cooled to ambient temperature and the prepared ACMNPs were separated by a permanent magnet and washed with water and 50% (v/v) water/ethanol, respectively, and stored under 20% (v/v) ethanol in 4 ◦C. 2.4. Activation and functionalization procedures for ACMNPs For partial crosslinking of the agarose particles and their functionalization by IDA ligand, an epichlorohydrin activation method was used as reported elsewhere [24]. After activation, 1 mL of the ACMNPs was added to 10 mL 0.1 mol L−1 ligand solution (pH 10, adjusted by 1 mol L−1 NaOH). The mixture was then shaken for 24 h at 30 ◦C. Finally, the particles were washed well with water and stored as a 20% v/v suspension under 20% (v/v) ethanol in 4 ◦C for later uses. Fig. 2 shows an schematic representation of the preparation procedure for ACMNPs and their activation and functionalization by IDA.