Functionalised magnetic markers

Today magnetic markers are widely used in biotechnological applications. They were originally developed by DYNAL BIOTECH to segregate specific biomolecules from a given solution [114]. The magnetic markers (also known as particles or beads) are specifically functionalised to bind to the target analyte and mixed with a solution. After the biomolecules are bound to the markers, the markers are removed from the solution by using a magnet. Because this method proved very successful, many companies offer a wide range of different magnetic markers. The commercially available markers differ in size from several nanometer to a few microns and in composition from pure Co to magnetite particles enclosed in different matrix materials.

Figure 1.7: Sketch of a typical magnetic marker

For choosing the most suitable particles from this wide variety, the requirements of the experiments have to be clear. The experiments presented in this thesis demand several attributes for the beads. First of all, the particles should have a high magnetic moment to be able to apply high forces to the beads. But because we want to manipulate single beads, they are not allowed to cluster. Therefore, the beads must be superparamagnetic and can't be ferromagnetic. Second, the particles are not allowed to be smaller than half the wavelength of light ($\lesssim$ 300nm), because they are tracked with an optical microscope. Third, the particles must not bind unspecifically to the sample surface (mostly SiO$_2$). This is especially important for all bond-force measurements. If there would be unspecific bonds to the surface, it would always be unclear if the rupture force corresponds to the bond in question, or just to an unspecified bond. Therefore, we tested 12 different magnetic markers on several surfaces in order to find out, if they adhere to the sample surface or not (confer section 4.4).

There are still many different commercially available beads that comply with these requirements. Figure 1.7 presents a sketch of a commonly used magnetic marker. The markers consist of superparamagnetic material enclosed by a matrix material and are functionalised on the outside with biomolecules.

According to the requirements stated above, three kinds of particles from three different companies are chosen. One might think that some matrix materials adhere to the surfaces and others don't. Our tests do not support this assumption, and so all three kinds of particles have a different matrix material. The beads used in this thesis have polystyrene, polyvinyl alcohol or silicate matrices. The magnetic material of all used markers is magnetite (Fe$_3$O$_4$). The biomolecules on the outside can be chosen as needed. For the bond-force measurements presented in chapter 4, we used beads functionalised with avidin or streptavidin.

Table: Properties of the magnetic particles used in this thesis [22,115,92]. The magnetic moment $m$ at 100Oe is measured with AGM, see section 2.6. (n.s.: not specified)

Company	Chemagen	Seradyn	Micromod
Product No.	M-PVA SAV1	30152104011150	39-18-153
Matrix material	polyvinyl alcohol	polystyrene	silicate
Magnetic material	Fe$_3$O$_4$	Fe$_3$O$_4$	Fe$_3$O$_4$
Diameter in $\mu$m	1	0.779	1.5
Particle density	n.s.	1.5g/ml	4g/ccm
Share of magn. mat.	50-60%	40%	n.s.
Magnetic moment $m$	1.82fAm$^2$	0.88fAm$^2$	0.4fAm$^2$

Table 1.1 presents the main properties of all three types of magnetic particles that were used for the bond-force measurements. The mean diameter ranges from 0.8$\mu$m to 1.5$\mu$m, which can be verified by Scanning Electron Microscopy (SEM) imaging. Figure 1.8 shows SEM images of all three particle types that were used for the bond-force measurements. The markers of one kind do not always have the same size (see e.g. figure 1.8b), and all three types have different surfaces. While the MICROMOD particles seem to have a very slick surface with other substances intermixed inbetween the beads, the CHEMAGEN particles have a more fleecy surface and there is nothing else in the solution. One possible reason is that the different matrix materials of the particles have a different contrast in the SEM. Other possible reasons are a different amount of biomolecules attached to the markers or a different method to attach the biomolecules.

Figure 1.8: SEM images of three different kinds of magnetic particles

[Micromod particles.] [Chemagen particles.] [Seradyn particles.]

Although the micron sized magnetite particles work well for the presented experiments, there is still room for improvements. Using nanoparticles instead of micron sized particles would greatly improve the binding capacity. Smaller markers would also reduce interfering effects in the behaviour of the biomolecules. But the magnetic moment should still be as high as possible. Single domain particles made of Co, FeCo or FePt with a diameter of only a few nanometers would meet these requirements, and such particles were already manufactured, e.g. by Hütten et al.  [68]. But there are still several problems with such small markers. For example, Co particles are probably not biocompatible and need, therefore, some coating for the use in biological systems. Furthermore the functionalisation of small metallic particles is not trivial, especially because the surface properties change the particle properties, too. So for these nanoparticles, there is still a lot of research to be done.