Editing Magnetic beads Preparation Methods (section)

== Silica coated superparamagnetic microbead ==

[https://www.hindawi.com/journals/jnm/2016/1730403/ https://www.hindawi.com/journals/jnm/2016/1730403/]

Tetraethylorthosilicate (TEOS), ammonium hydroxide (NH4OH), styrene, 2,2′-azobis-isobutyronitrile (AIBN), dibenzoyl peroxide (BPO, including 97% of active compound), dibutyl phthalate (DBP), sodium silicate, Fe3Cl3·6H2O, FeCl2·4H2O, ethylene glycol, N,N-dimethylformamide (DMF), sodium dodecyl sulfate (SDS), polyvinylpyrrolidone-40 (PVP-40, Mr: 40,000), N,N-diisopropylethylamine (DIEA), and (3-aminopropyl)triethoxysilane (APTS) were purchased from Sigma-Aldrich (MO, USA).

2.2. Methods 2.2.1. Preparation of Monodisperse Macroporous PS-DVB Beads Monodisperse macroporous PS-DVB (polystyrene-divinylbenzene) beads were prepared by seeded polymerization methods. Monodisperse PS seeds (4 μm) were prepared using dispersion polymerization method [49]. Dispersion medium was ethanol/2-methoxyethanol (3 : 2) which contains 1 g of PVP-40 as a steric stabilizer (90 mL). AIBN (150 mg) was dissolved in styrene (15 mL) in which inhibitor was removed and then was added to the as-prepared dispersion medium. After sonication for 10 min, dispersion polymerization was performed in a cylindrical reaction chamber with shaking (120 cpm) at 70°C for 20 h. The suspension was centrifuged and the precipitates were washed intensively with distilled water by repeating centrifugation and vortexing. Finally, the PS seeds were washed with ethanol and vacuum-dried overnight affording PS seeds (4 μm, 8.3 g).

Swelling and polymerization processes were performed in a glass reactor equipped with overhead stirrer and a reflux condenser in an oil bath equipped with a temperature controller. The two-step seeded polymerization method was employed for preparation of monodisperse macroporous poly(styrene-co-DVB) (PS-DVB) beads. The PS seeds (4 μm, 700 mg) were dispersed in DBP (0.7 mL) emulsified aqueous medium (100 mL) containing 0.25% (w/w) SDS. The resultant dispersion medium was stirred (400 rpm) at room temperature for 20 h for swelling the PS seeds with DBP. Mixture of styrene (4.6 mL) and DVB (2.3 mL) in which BPO (240 mg) was dissolved was emulsified in an aqueous medium (100 mL) containing 0.25% (w/w) SDS by using homogenizer for 1 min. The emulsified monomer solution was added to the DBP-swollen PS seeds dispersion medium being stirred. Monomer swelling was performed for 20 h at room temperature with continuous stirring (400 rpm). After swelling process, an aqueous solution of 10% (w/v) PVA in deionized water (10 mL) was added to the dispersion medium and the medium was purged with nitrogen stream for 30 min. The seeded polymerization was performed at 70°C for 20 h with continuous stirring (200 rpm) to obtain monodisperse PS-DVB beads. The polymer beads were intensively washed with warm deionized water (50°C) by repeating washing and centrifugation. Subsequently, the collected beads were washed with ethanol and THF intensively to remove DBP and linear polymer. Finally, the beads were dried in vacuum at 30°C for 24 h to obtain macroporous PS-DVB beads (7.5 μm, 2.5 g).

2.2.2. Sulfonation of Macroporous PS-DVB Beads Sulfonation of the PS-DVB beads was performed with reference to the reported method [50]. Briefly, monodisperse PS-DVB beads (1 g) were added to 5 mL of acetic acid in an ice bath. Sulfuric acid (50 mL) was then slowly added to the beads at room temperature and the temperature was increased up to 90°C and the resin mixture was stirred for 30 min to 2 hr. The dispersion was poured into iced water (400 mL) to quench the reaction and the sulfonated PS-DVB beads were collected by centrifugation. The beads were extensively washed with deionized water by repeating centrifugation-decantation. Subsequently, the sulfonated microspheres were washed three times with ethanol and dried under vacuum (1.1 g).

2.2.3. Magnetization of Sulfonated Macroporous PS-DVB Beads The sulfonated macroporous PS-DVB beads (500 mg) were dispersed in deionized water (10 mL) at room temperature with mechanical stirring (200 rpm) and purging with nitrogen. A freshly prepared mixture of FeCl3·6H2O (618 mg, 2.26 mmol) and FeCl2·4H2O (257 mg, 1.28 mmol) in deionized water (10 mL) was added to the dispersion for adsorption. After 2 h, with continuous stirring 28% ammonium hydroxide (50 mL) was added dropwise to the beads suspension for 40 min. The magnetized microspheres were isolated from the mixture by centrifugation and briefly washed with 25% TFA and then extensively with deionized water and ethanol. Finally, the magnetized microspheres were dried under vacuum (magnetized microspheres, 7.5 μm, 653 mg).

2.2.4. Preparation of Silica Coated Magnetic Beads APTS solution (1% (v/v), 100 mL) was added to 100 mg of magnetic sulfonated PS-DVB beads and was shaken for 10 min at room temperature. Thereafter, ammonium hydroxide (28%, 2 mL) was added to the bead dispersion and was shaken for 20 min at room temperature. To the dispersion TEOS (2 mL) was added and vigorously shaken for 12 h at room temperature. The resulting beads (silica coated magnetic beads) were collected by magnet and washed with ethanol five times.

2.2.5. Streptavidin Separation of Silica Coated Magnetic Beads APTS (240 μL) and ammonium hydroxide (40 μL) were added to 4 mL absolute ethanol containing 200 mg of silica coated magnetic beads and incubated for 6 h at room temperature. The resulting beads (amine-silica coated magnetic beads) were collected by magnet, washed with ethanol five times, and dispersed into PBS, pH 7.2, to obtain 10 mg/ml of amine-silica coated magnetic beads.

Biotin (5 mg) was activated with EDC (40 mg) and NHS (20 mg) in PBS, pH 7.2, for 1 h at room temperature. Then, this solution was added into amine-silica coated magnetic beads (1 mg, 100 μL) to modify biotin on its surface. The biotinylated silica coated magnetic beads were collected by magnet, washed with ethanol five times, and dispersed into 100 μL PBS, pH 9.0. The biotinylated silica coated magnetic beads were passivated with 5% BSA in PBS, pH 9.0, for preventing nonspecific binding.

Streptavidin-FITC (100 μg) in PBS pH 9.0 was added into the biotinylated silica coated magnetic beads and incubated for 30 mins at room temperature. The biotinylated silica coated magnetic beads after incubation with streptavidin-FITC were collected by magnet, washed with PBS, pH 9.0, containing 0.1% Tween 20 for five times, and dispersed into 100 μL PBS, pH 9.0. The fluorescent images were collected by the Olympus BX-UCB at room temperature with 200 ms exposure.