Editing Magnetic beads Preparation Methods (section)

== iron oxide silica particles ==

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854921/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854921/]

iron oxide silica particles To synthesize the Fe3O4@Silica for RNA extraction, the following chemicals of analytical reagent grade were received and used without further purification. Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), styrene, glycidyl methacrylate (GMA), divinylbenzene (DVB), azobisisobutyronitrile (AIBN), polyacrylic acid, guanidine hydrochloride (>99%) and ethanol anhydrous were obtained from Sigma Aldrich. Ammonia in water 28–30% w/w, sodium citrate tribasic dihydrate, nitric acid (90%) were obtained from BDH. Tetraethyl Orthosilicate (TEOS), 3-glycidyloxypropyltrimethoxysilane (GTMOS), (3-Aminopropyl)triethoxysilane (APTES) were purchased from TCI (USA). All aqueous solutions were prepared with deionized water obtained from a Milli-Q Integral system (resistivity = 18 MΩ cm, EMD Millipore). Zymo MagBinding® Beads was obtained from Zymo Research, USA for comparison in RNA binding. Dynabeads® MyOneTM, M280 from Thermofisher were used as the microscopic reference sizing materials, employed at 800x magnification.

2.2. Synthesis of Fe3O4@Silica from iron oxide nanoparticles (Fe3O4) as seeds Fe3O4 nanoparticles were prepared by the basic co-precipitation method. 4.46 g (16.6 mmol) FeCl3·6H2O and 1.6 g (8.1 mmol) of FeCl2·4H2O were dissolved in 80 ml of deionized water at room temperature. The solution was degassed by bubbling with Ar for 5 min and magnetic stirring in conical flask. 10 ml of ammonia 28–30% w/w was added dropwise to the solution and stirred for another 10 min. at room temperature. Following with heating for 1 h at 90 °C, iron oxide magnetite (Fe3O4) formed was cooled down to room temperature and magnetically separated, washed with deionized water until pH 8 reached.

Fe3O4 particles formed in above method were treated with 2 M nitric acid for 5 min with stirrer mixing at room temperature, we believe this treatment can stabilize the Fe3O4 particle surface [25, 26]. After the reaction, the particles were washed with deionized water and magnetically separated until the pH reached ∼2.5. Then, 10 ml aqueous solution of 0.5 M sodium citrate was added to the 30 ml particles suspension. The resulting solution was stirred for 1 h at room temperature to prepare the surface for silica coating. Finally, the citrate surface stabilized particles were magnetically recovered, washed thoroughly with deionized water and freeze-dried. Dried beads weighed 1.77 g (MW 231.53, 7.6 mmol); yield = 88%. Dynamic light scattering (DLS) and zeta potential measurements were obtained using a Malvern ZETASIZER NANO ZSP. The sample concentrations were adjusted to ∼0.1% w/v particles in deionized water. The samples were sonicated in ultrasonic bath for 1 min and pre-equilibrated for 3 min at 25 °C prior to each measurement. Zeta potential measured was −28.5 mV. Scanning Electron Microscopy (SEM) analysis was obtained from FEI Helios Nanolab 660. SPION stabilization with citrate is a protocol inspired by Khosroshahi et al and Digigow et al [25, 26]. This 1-pot, 2-step procedure leads to citrate surface-stabilized Fe3O4 nanoparticles that can be stored in dry form at 4 °C.

To prepare the silica glass surface of the above citrate- Fe3O4 particles. Re-suspension and sonication of these dry particles in 10% v/v water in ethanol, followed by silica shell formation with hydrolysis of tetraethoxysilane (TEOS) under alkaline condition using 28–30% ammonia, results in silica magnetic particles that can be magnetically separated without further purification (Fig. 1). At the end, the silica coated magnetic microparticles were rinsed with ethanol and dried in vacuum. Dried microparticles weighed 3.4 g.

Different silica mass ratio were obtained by using different reactants ratio of TEOS:NP, ranging from 2:1, 4:1 to 6:1. Corresponding mass ratios of silica coating ranged from 45%, 55% and 62% were obtained. A mass ratio of TEOS vs starting particles NP of 1:1 was not pursued because of the possibility of incomplete silica enclosure on the Fe3O4 magnetic particles. And we observed that a higher silica mass ratio than 55% was not preferred as the additional weight of the silica shell resulted in a slower magnetic response of the coated Fe3O4@Silica particles.

2.3. Formulation of Fe3O4@Silica for RNA extraction in Zika assays Preparation of 10% w/v of the Fe3O4@Silica in 1 M Gu.Cl buffer for Zika assays was done by suspension of 100 mg of the Fe3O4@Silica prepared above in 500 μl deionized water, then added with 2 M Gu.Cl buffer to make up to 1.0 ml final volume. Zeta potential of this formulated Fe3O4@Silica was measured as −4.84 mV.

2.4. Alternative methods for synthesis of Fe3O4@Silica from polymer seeds Alternative methods to prepare Fe3O4@Silica beads were also tested; for example, polymerizing a mixture of (1) TEOS with γ-aminopropyl-triethoxysilane (APTES); (2) TEOS with (3-mercaptopropyl)trimethoxysilane (MPTMS); and (3) amino surface of magnetic polystyrene particles reacted with (3-glycidyloxypropyl)trimethoxy silane (GLYMO). In typical examples, in-house synthesized amine surface polymer microparticles with approximate 2 μm was used as seeds. 0.5 g of seeds were reacted with 0.2 ml 3-glycidyloxypropyltrimethoxysilane (GTMOS) for 1 h at room temperature in 5 ml ethanol, followed with 0.1 ml tetraethyl orthosilicate (TEOS) or a mixture of 0.1 ml tetraethyl orthosilicate (TEOS) and 0.2 ml (3-Aminopropyl)triethoxysilane (APTES) in 0.2 ml ammonia 28–30% w/w for 4 h at 90 °C (Fig. 3). After reaction, the magnetic particles were magnetically separated and washed with ethanol (5 times) with centrifugation at 3000 × g or separated magnetically. Zeta potential measured microparticles with silica surface formed from (TEOS:APTES, 2:1, v/v) = +39.2 mV microparticles with silica surface formed from TEOS only = +15.3 mV. This preliminary study on the ratio of TEOS:APTES relationship with surface Zeta potential demonstrates the fine tunable controls on the surface charges by the design of combination of silylating reagents used.