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Magnetic bead separation is a powerful tool for the separation, isolation and extraction of nucleic acids, cells and proteins. Magnetic bead separation is commonly applied in next-gen sequencing (NGS), PCR, qPCR, protein and peptide purification, immunoassays, cell separation, pathogen detection, exosome isolation.
In order to bond to their target molecule, magnetic beads must be coated with a ligand that will attach to the target. Antibodies and nucleic aptamers are commonly used as ligands and allow for an easily tunable target. There are other alternatives such as the ThermoFisher dynabeads which are coated with streptavidin layer that bonds to biotinylated ligand probe molecules which then attach to the target molecule.
In order to use magnetic beads to separate, isolate and extract nucleic acids, cells and other proteins from a solution, the beads must have two requirements.
Superparamagnetic beads meet both of these criteria. If iron oxide beads, which would normally be considered ferromagnetic, have a particle size of 3-50nm then the entire bead will consist of only a single magnetic domain. This essentially removes the hysteresis or permanent magnetization typical of a ferromagnet. What you’re left with is a bead that has the high strength of a ferromagnet but is only magnetized in the presence of an external magnetic field. This is called a superparamagnetic bead and is ideal for magnetic bead extraction.
Short Answer: They don’t clump together because superparamagnetic particles are only magnetized in the presence of an external field.
Magnetic beads need to be able to be mixed in a solution, and then pulled to the side with a magnetic field. If we used a normal magnetic particle, the magnetic attraction between particles would cause them to clump together and never mix in the solution.
What we need is a material whose magnetic moment can be turned off for mixing, and turned on for extraction. One solution would be to use a material with paramagnetic properties. These types of materials become magnetized in the presence of an external magnetic field, but lose their magnetization if the external field is removed. This means they could mix well in the solution, and then be pulled to the side by putting a magnet on the side of the reservoir. Unfortunately, paramagnets have a low magnetic susceptibility and only become weakly magnetized. Using them for an extraction would take a very very long time.
An ideal solution would be a particle that has the strength of a permanently magnetized ferromagnet, but the on/off aspect of a paramagnet. Luckily, it was discovered that very small iron oxide particles are superparamagnetic. When these particles are nano-scale their magnet moments become randomized and cancel each other out. However, the individual particles still retain their magnetic moments and an external magnetic field will cause them to move just like any other magnet. Furthermore, they retain the high magnetic susceptibility of iron oxide and are strongly magnetic.
Superparamagnetism is an effect that occurs in ferrous particles whose particle size is less than the size of a single magnetic domain and typically happens around 3-50nm depending on the material. Since the particle has a single magnetic domain, the magnetic moments of every atom will combine into one large magnetic moment. The particle will typically have two antiparallel magnetic states and will switch between them randomly. Basically, the north and south poles of the particle will randomly switch back and forth. The mean time for this switch is called the Neel Relaxation time.
If the Neel Relaxation time is sufficiently lower than any measurement interval then it will appear as though the particle is not magnetic at all. If you consider two superparamagnetic particles floating in space, they will be attracted to each other when a north pole faces a south pole, and repelled when a north/south pole faces a north/south pole. As the magnet moments flip back and forth the particles will be repelled as often as they are attracted and there will be no net motion. For all intents and purposes they behave as though there was no magnetic force at all.
In the presence of an external magnetic field a superparamagnetic particle will be biased towards one of its antiparallel magnetic states and will stop switching between them or, more precisely, it will spend proportionally more time in it’s biased state depending on the strength of the external field and the temperature of the particle. This means that it will behave as you would expect a normal ferromagnetic particle to, but only in the presence of a magnetic field.
In summary, superparamagnetic particles can be magnetized as strongly as a ferromagnet, but lose their magnetization in the absence of an external magnetic field.
A Halbach array is an array of magnets arranged so that one side has a strong magnetic field and the other has a weak magnetic field. By arranging the magnets in alternating vertical and horizontal directions, the horizontally aligned magnets will reinforce the north and south poles on one side of the array while diminishing the north and south poles on the other side. In the figure you can see a Halbach array designed to have a strong magnetic field above the array and a weak field below.
ClickBio uses this effect to enhance the magnetic field in our XBase96PCR magnet. This pattern of magnets allows for an extremely strong magnetic field that doesn’t extend very far above the magnet. This is ideal for the form factor of a PCR microplate and can greatly reduce magnetic bead extraction times with 96 well PCR plates.
