The coating and surface chemistry of magnetic beads governs the binding efficiency of target DNA or RNA. Superparamagnetic beads for life science applications come in two general forms: core-shell type and embedded type. The core-shell synthesis method produces beads composed of a single superparamagnetic core with a polymer or silica surface coating. One example is a bead composed of a magnetite core surrounded with a dextran shell. Other beads of the core-shell type are composed of a polystyrene or polyvinyl alcohol (PVA) core surrounded by superparamagnetic particles and protected by a surface coating.
Sometimes these core-shell beads can have multiple layers of superparamagnetic particles alternating with encapsulation material. An alternative method, called embedding, produces superparamagnetic beads composed of a monodisperse matrix such as polystyrene, agarose or sepharose impregnated with multiple 10 nm iron-oxide nanoparticles (magnetic pigment). These beads are typically hundreds of nanometers in diameter and are sealed with a material that prevents loss of the magnetic pigment.
Regardless of bead type, synthesis method, or core composition, the surfaces of the superparamagnetic beads are chemically modified to allow adsorption of nucleic acids to their surfaces. Any nucleic acid adsorption chemistry is ideally reversible to allow removal of magnetic particles following the extraction procedure. This is necessary because the presence of magnetic particles in the nucleic acid isolate could interfere with downstream qPCR or sequencing methods.
Reversible nucleic acid adsorption systems
Silica-coated magnetic particles have a reversible binding affinity that is dependent on salt concentration. The DNA binds under high-salt conditions, and is released under low-salt conditions. The mechanics of DNA adsorption to silica is not immediately obvious. From a purely electrostatic viewpoint, DNA should be repelled from silica because they both have negative surface charge. The DNA and the silica each have a Debye double layer. The Debye double layer represents the distance that it takes for the electrostatic potential to decay. It is a function of ionic concentration: the higher the salt concentration, the thinner the double-layer. The idea is that DNA and silica can move closer together in high salt solutions and attractive Van der Waals forces overcome repulsive electrostatic forces. Also, lowering the pH decreases the negative charge density on the surface of the silica, which may reduce the repulsive force between DNA and silica. Following magnetic separation the nucleic acids are released under low ionic conditions.
) carboxylated surface
Most paramagnetic beads can be functionalized with a surface coating of carboxyl groups. DNA reversibly binds to the surface of these beads in the presence or absence of polyethylene glycol (PEG) in varying salt concentrations. In a similar mechanism to that of silica coatings, the concentration of PEG and salt is very important to the success of this strategy; the electrostatic potential must be overcome to allow attractive Van der Waals forces to prevail.
c) amine and imidazole moieties
Amine groups and imidazole molecules are sensitive to changes in pH. They have a surface charge of zero in neutral solution, but are positive in acidic solution and negative in basic solution. The negatively charged nucleic acid adsorbs to the positively charged amine or imidazole moiety in acidic solution. After magnetic separation the DNA or RNA can be recovered by increasing the pH to make the solution more basic. Desorption of DNA from the imidazole group is most efficient at 80°C, which indicates that other binding strategies are involved other than only electrostatic attraction and repulsion. Amine modification is typically used in conjunction with silica coatings. One magnetic particle commonly used for nucleic acid purification is an amino-modified silica-coated magnetic nanoparticle (ASMNP). Recent research explained in more detail in chapter 7 suggests that the imidazole modification may perform better at reversible adsorption of DNA than ASMNPs.
Overall, the reversible DNA adsorption chemistry on the surface of superparamagnetic beads is simple and highly reliant on environmental conditions. The silica, -COOH and -NH2 functional group chemistry is well-understood and easily manipulated by ionic strength, pH, and temperature. This allows for a high degree of control over DNA adsorption and desorption from the bead surface.