Magnetic Separation

Magnetic particles have been used as support for the separation, selective isolation, and purification of molecules [32]. For example, in biomedical diagnostics, they can replace the cumbersome steps of centrifugation or filtration [32-34]. The major techniques all involve chemical grafting of biomolecules onto magnetic beads to target specific separation of captured biomolecules or of analytes [29]. Since magnetic supports can be separated from solutions containing other species (e.g., suspended solids, cell fragments, and contaminants), magnetic affinity separation is useful for crude samples [35]. Various magnetic particles can be adapted to these kinds of applications, including large particles (above 1 pm) [29], silica magnetic particles [32], and nanoparticles (below 100 nm).

Therefore, use of a magnetic field to separate composite magnetic particles is, compared with the alternatives, very simple, often cheaper, and above all faster. For example, bacterial control in the food industry requires 10 min of magnetic particle use instead of the 24 h required with the traditional methods of analysis because the target bacteria concentrations are too weak to be characterized and require culturing. Fast magnetic separation results in direct concentration of the bacteria and therefore eliminates this slow step. The need to apply a powerful magnetic field may, however, be quite expensive, depending on the particle properties. Regardless of the use considered, magnetic particle carriers must have the following properties:

• Colloidal and chemical stability in the separation medium

• Nonmagnetic remanence

• Does not release of iron oxide during biomedical applications

• Low sedimentation velocity compared with magnetic separation

• A surface that is biocompatible with the relevant biomolecules

• Allows complete, rapid, and specific separation

Two specific methods are frequently used in biomedical diagnostics for detecting disease with magnetic particles and specific interaction between biological molecules (e.g., antibody/antigen or nucleic acids):

1. Direct separation. The biological sample containing the target molecules is mixed with magnetic particles bearing specific antibodies (generally called sensitive particles). After incubation under given conditions (time, buffer composition, temperature) and the subsequent immunological reaction between the immobilized antibody on the particle and the target antigen, the magnetic particles are separated by applying a magnetic field with a single magnet. Such direct capture (Fig. 1) and separation can also be performed with two kinds of antibodies. In this case, the first antibody is fixed on the colloidal particle as a spacer arm for immobilization of the second antibody for specifically capturing the target. The first antibody favors the orientation of the specific antibody immobilized via its Fc part onto the spacer arm-like antibody.

2. Indirect separation. The target molecule in a biological sample is first recognized by a specific antibody capable of reacting with the second antibody, which has been chemically grafted onto the surface of magnetic colloidal "carrier" particles. This method requires a thorough knowledge of molecular biology. These indirect methods are often more specific than the direct binding of antibodies [36] (Fig. 2).

On the physical level, the separation of magnetic particles as a function of the magnetic field can be explained and discussed basically on the basis of

FIG. 1 Schematic illustration of direct specific separation of a target molecule.
Illustration Immunomagnetic Separation

FIG. 2 Schematic illustration of indirect specific separation of a target molecule.

Biological sample

FIG. 2 Schematic illustration of indirect specific separation of a target molecule.

forces acting (magnetic force Fm and electroviscous force Fv) on the magnetic particles placed under the magnetic field (H), as summarized in the following expression (at equilibrium state):

Fv is the friction force (or electroviscous force), which is basically the resistance to the displacement of the particle in a liquid medium; it is expressed as a function of the medium viscosity by Stokes' equation: -6 rcqRV, where R is the hydrodynamic radius of the particle, n is the viscosity of the medium, and V is the separation velocity.

Fm is the magnetic force due to the applied magnetic field (magnetic attraction force) and can be expressed as a function of various parameters:

where H is the magnetic field intensity, VH is the gradient of the magnetic field H, | is the magnetic susceptibility (| = |0 • Ms4rcR3/3), Ms is the saturated magnetization of the colloidal magnetic particles (principally related to the nature of iron oxide used), and | 0 is the permittivity of the vacuum.

The separation speed (V) can therefore be expressed from the previous equations and as a function of saturated magnetization:

Consequently, the separation speed increases with both the radius of the particle and the saturation of the magnetization. As expressed by the above equation, the saturated magnetization (Ms) is proportional to the content of magnetic material. In a given medium and in the presence of a fixed magnetic field, separation speed (V) therefore depends on the iron content, the magnetic properties of the particles, and the hydrodynamic size of the final microspheres. The above equation can be used to illustrate the parameters that affect the magnetic velocity of the particles under the magnetic field applied.

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