Background

The ability to achieve solid-phase reversible immobilization (SPRI) (DeAngelis et al., 1995) is one of the most useful characteristics of functionalized paramagnetic nanoparticles (MNPs). Together with their scalability and potential for automation, MNPs are ideal candidates for the development of high-throughput protocols regarding nucleic acid purification (Hawkins et al., 1994; DeAngelis et al., 1995; Rohland and Reich, 2012). The small nano- or microparticles are able to reversibly bind nucleic acids under dehydrating conditions and can be safely immobilized by a strong magnet during consecutive wash or manipulation steps. Bead-based protocols can be adapted easily for multi-well formats, allowing the processing of hundreds of samples simultaneously. Additionally, MNPs are very cost-effective as the beads can be obtained very cheaply, both commercially and self-made. Although their advantages over more established column-based methods are obvious, surprisingly little effort has been put in the development of open-source protocols featuring paramagnetic beads.

Protocols for the synthesis of ferrite nanoparticles have been published before (reviewed in Houshiar et al., 2014; Singh et al., 2014; Wu et al., 2015; Majidi et al., 2016). We adopted the commonly used co-precipitation method due to its efficient and robust performance and the fact that it does not require any specialized equipment (Choi et al., 2007). For this, a solution of FeCl₂ and FeCl₃ in a 1:2 molar ratio is prepared and slowly dripped into a preheated NaOH solution. This forms a black precipitate consisting of Fe₃O₄ particles with an approximate diameter of 5 to 20 nm as judged by electron microscopy (Figure 1A). As oxygen is known to interfere with ferrite precipitation reactions, the NaOH solution should be degassed and/or preheated to 80 °C or higher before adding the iron solution. After the synthesis reaction, the nanoparticles are washed multiple times with deionized water. We recommend starting the respective coating reaction immediately after the synthesis as the particles are prone to oxidation. However, it is possible to stabilize them using various chemicals, such as detergents, sodium oleate or polyvinylpyrrolidone (PVP) (reviewed in Singh et al., 2014; Majidi et al., 2016). Upon lyophilization, the core MNPs can be stored in an air-tight container under inert atmosphere.


Figure 1. Synthesis of BOMB paramagnetic beads. A. Equipment setup for BOMB MNP synthesis. Place a magnetic stir bar in both the water bath as well as the round bottom flask in order to avoid heat accumulation. B. Synthesized and washed paramagnetic core particles. C. Paramagnetic core particles in transmission electron microscopy (TEM). D. Silica-coated paramagnetic beads in scanning electron microscopy (SEM) and TEM. E. Carboxyl-coated paramagnetic beads in light microscopy (LM), with and without an applied magnet.

Encasing of the ferrite nanoparticles in a silica coat prevents oxidation and leakage of iron ions and provides an inert surface for nucleic acid precipitation without the risk of irreversible binding. The silica deposition reaction outlined here is based on a modified version of the Stöber method (Stöber et al., 1968). It utilizes tetraethyl orthosilicate (TEOS) which is hydrolyzed in a basic environment, leading to the deposition of a SiO₂ layer surrounding the paramagnetic core. Conveniently, the thickness of the silica coat, and therefore the size of the beads, can be controlled by the amount of TEOS added to the reaction (Kim et al., 2007), allowing a precise adjustment depending on the respective downstream application. The provided standard coating protocol yields silica-coated beads with an average diameter of approximately 400 nm (Figure 1B), which perform well for a wide range of nucleic acid purification and manipulation experiments from bacteria, mammalian cells, tissues (Figure 2) and many more (Oberacker et al., 2019).


Figure 2. BOMB nucleic acid extractions. Data and figures are taken from Oberacker et al. (2019). A. Total DNA and plasmid extracted from E. coil. MW: GeneRuler DNA Ladder Mix (Thermo). B. TNA, DNA and RNA isolation from HEK293 cells. MW: GeneRuler DNA Ladder Mix (Thermo). C. gDNA isolated from various rabbit (O. cuniculus) tissues. MW: Hyperladder I (Bioline).

As an alternative to a silica-coat around the ferrite particles, a layer of a carboxyl-modified polymer can provide stability as well as a surface with a weak negative charge, thus altering the electrostatic interaction with nucleic acids and consequently the functionality of the beads (Figure 1C). Here, we use methacrylic acid (MAA) monomers, which form a layer of polymethacrylic acid (PMAA) around the ferrite core particles during a free-radical retrograde precipitation polymerization reaction (Aggarwal et al., 1996). Potentially, carboxyl- and silica-surfaces can be further modified to enable a broad range of additional functionalities to the paramagnetic beads.