Results

All bond-force measurements are viewed with an optical microscope and recorded with a CCD-camera system (see section 2.7 for more information about the setup). Figure 4.7 shows three images of such a video where two rupture events are recorded. The current is slowly increased during the measurement and two beads still bind to the biotinylated surface at a current of 73mA (a). One second later, at a current of 74mA, the upper marker is gone, so the bond was ruptured (b). Several seconds later, at a current of 81mA the bond of the second marker was ruptured (c).

Figure 4.7: Three images of a recorded video. At 73mA two streptavidin markers still bind to the biotin (a). At a current of 74mA, the upper marker is ruptured (b), and at 81mA the lower is ruptured (c). See CD for the complete video.

The recorded videos of all measurements were evaluated for such rupture events (see CD for videos of all rupture events). In order to calculate the bond-force $F_{\rm mag}$ with equation 1.10, we need three variables. We get the current $I$ and the distance $R$ directly from the recorded video, and together with the magnetic moment $m$ of the markers (see table 1.1 on page ), the magnetic force for every rupture event is calculated.

Figure 4.8 presents the results of all bond-force measurements with CHEMAGEN beads (a) and MICROMOD beads (b). The measured bond-forces for the streptavidin-biotin bond (a) are very low (in the range of 25-475fN), and the distribution clearly shows two maxima. The average of the first maximum is at a bond-force of 55.9fN ( $\sigma_{\bar x} = 18,9$fN) and the average of the second maximum is at 244,7fN ( $\sigma_{\bar x} = 64,5$fN). There is also a single event at 457fN, which will be discussed later. The bond-forces of the avidin bonds are even lower in the range of about 10-90fN. Again, there are two maxima in this distribution, but they are not clearly separated. The first maximum (in the range of 0-40fN) is at 15.9fN ( $\sigma_{\bar x} = 2,2$fN) and the second maximum (in the range of 40-90fN) is at 58,4fN ( $\sigma_{\bar x} = 5,0$fN). It is apparent that for both bonds, the second maximum is about 4 times higher than the first maximum, which dashes the idea that this is just a double bond. A detailed explanation will be given below.

Figure 4.8: Distributions of the measured bond-forces for streptavidin-biotin (a) and avidin-biotin (b) bonds.

[streptavidin-biotin bond] [avidin-biotin bond]

The streptavidin-biotin bond was tested additionally with markers from the SERADYN company (confer section 1.2). These markers did not bind very well, and therefore, only very few events could be measured (see the CD for all rupture events with the SERADYN markers). The mean value of these events is quite near to the first maxima of the rupture events with CHEMAGEN beads, at 57,6fN ( $\sigma_{\bar x} = 47,8$fN).

The width of the maxima in these distributions is mainly caused by the distribution of the magnetic moment of the markers. The magnetic moment can only be measured for hundreds or thousands of beads, and then the average over all beads is calculated. Because the beads have a wide distribution in size, the magnetic moment has probably a similarly wide distribution.

In order to investigate the streptavidin-biotin bond even more with this method, we tried to put the system upside down. The streptavidin was put on the surface, where it can bind to gold, and biotinylated magnetic markers were used on top. Unfortunately, the biotinylated markers already bound directly to the gold surface without any streptavidin. Therefore, one major necessity of the experiments wasn't met, and the upside-down system could not be used.

The number of bonds between a bead and the surface depends on the concentration of the biotinylated oligonucleotides on top of the surface. During the first experiments, a very high concentration was used and, therefore, nearly all markers bound to the surface and no marker could be ripped of. Of course, the reason for this was that the markers had several full bindings and, therefore, couldn't be ruptured. When no biotinylated oligonucleotides are on the surface, of course no bonds at all occurred (confer section 4.4). For the bond-force measurements in this thesis, the best concentration of oligonucleotides is between 10 and 1000nM.