2.1 Binding force measurement using AFM
Atomic force microscopy (AFM) has been developed for few decades and used widely for scanning images in nano and micro scales. With a cantilever beam probe, AFM scans the sample surface by tapping or contacting and detects the force and the displacement of the cantilever beam. It then reconstructs the topography of the samples.
In 1994, Florin et al. used AFM to measure the specific interaction between biotin and avidin [18]. AFM probe with nitride tip was functionalized with avidin first. It then contacts with a biotinylated agarose bead and measures the force and the displacement of the beam, as shown in Fig 2-1. In Figure 2-1, the horizontal line means no forces acting on the cantilever beam as the tip approaches the surface. Suddenly, there is a compressive force acting on the tip when it contacts with the surface. In the retraction process, the adhesion force between bead and tip results in a deflection of the cantilever beam toward the bead. With increasing bend of cantilever, the tension force on the adhesive bond between bead and tip increases until the bond breaks. When the bond between biotin and avidin breaks, a sudden step occurs, which shows the maximum adhesion force between biotin and avdin in the retraction process. This condition allows only a limited number of molecular pairs to interact. The force required to separate the biotinylated agarose bead and the avidin coated tip was quantized in integers multiples of 160 ±20 piconewtons. In 1994, Lee et al. measured the force between two complementary DNAs using AFM [19]. As the reported results, the adhesive forces are 1.52, 1.11, and 0.84 nN, which are associated with 20, 16, and 12 base pairs of complementary DNAs separately.
Besides DNA and protein interaction, specific antibody and antigen interaction was investigated by Dammer et al. in 1996 [20]. In addition, primary and secondary antibody interactions and aptamers interactions were studied by Lv in 2010 [21] and Nguyen in 2011 separately [22]. Though a lot of different ligands and receptors have been discussed, they all had a same problem: it is hard to measure one pair of ligand and receptor directly at the same time. Always, there are multiple pairs of interactions between tip and sample surface in measurement because the tip of the probe is still too large compared to antibody, protein or DNA. However, it is still the most common way to measure the molecule interactions.
Displacement Force
Figure 2-1 Force and displacement relationship when AFM tip approaches to and retracts from the target protein [18].
2.2 Hydrodynamic shear assay
Though the interactions between ligands and receptors can be measured directly using AFM, there are some literatures which reported to measure the interactions by hydrodynamic shear assay because it needs expensive instruments and well controlled environment to reduce noises to measure the binding force using AFM. In 1990, Roberts et al. had tried to model the cell adhesion by conjugating beads to antibodies and then adhered them to surface-coated complementary antibodies. To investigate the interactions between two antibodies, they conducted beads detachment experiment in a radial flow device [23]. Schematic diagram of the radial flow device is shown in Figure 2-2. The fluid is injected into the radial discs through the tube in the middle of the device. Because the cross-sectional area of the flow increases with radial distance, the flow velocity and the shear force decrease. Therefore, within a circular zone around the inlet where the shear forces are higher, particles are swept away. However, within the outer zone where shear forces are smaller the beads can adhere. There is a boundary between these two zones, and the radius marked this boundary is defined as critical radius. The reported mathematical model interprets the experiment data and analyzes the adhesion between ligands and receptors. DNA binding was investigated with hydrodynamic flow in 2006 by Zhang et al. [24]. Similarly, beads coated with DNA specifically adhere to the surface with complementary DNAs; however, the whole device was a parallel plate flow chamber. The flow was controlled with a syringe pump, and the detachment experiment was observed with a converted microscope.
The interactions between the ligands and receptors were analyzed in these two literatures, but none of them had quantified the binding force. Lorthois et al.
investigated Fibrin/Fibrin-specific molecular interactions in 2001 [25]. With some
critical assumptions, Lorthois modeled the bond between fibrins as Hookean springs and calculated the binding force of the DNA pairs, suggesting that this force is about 400 pN.
Since microfluidics emerged in 1980s, precise control of small volume fluids makes it possible to reduce the cost and time of reaction in biological science. Further, the microscale fluids form stable laminar flow in microchannel naturally so the flow field can be easily controlled. With many advantages of microfluidics, a lot of researches have realized hydrodynamic shear assay in microfluidic systems.
Yokokawa et al. measured the adhesion force between kinesins and microtubule using hydrodynamic force produced by microfluidic flow in 2011 [26]. The microchannel is
Figure 2-2 Side view of radial flow device for detachment experiment [23].
fabricated by the poly(dimethylsiloxane) (PDMS) using casting method and held by aluminum plate holder. Microtubule is immobilized on the bottom of the channel, and the beads coated with kinesins adhered to it. Just like the literature above, the device was linked to syringe pump to perform detachment experiment. As the fluid force exceeded the adhesion force between kinesins and microtubule, the beads were swept away. In addition, the ATP was added into the fluid to decrease the binding strength between kinesins and microtubule. As the results, the binding forces are 362.9 pN and 31.3 pN for conditions of ATP absence and ATP presence separately.
There are same problems in hydrodynamic assay: there are multiple bonds between one bead and the surface. Binding density estimation may be a good solution. The contact area of the bead can be calculated; therefore, the bond numbers between bead and the surface can be estimated with binding density. Today, a lot of efforts are taken to measure single bond between ligand and receptor; however, no good methods can investigate the binding interactions with single bond.
2.3 Other methods to study the interaction between ligands and receptors
Except for the two methods mentioned above, there are other methods to study the interactions between specific ligands and receptors, such as unbinding cells by two micropipettes, optical tweezers method, centrifugation method and magnetic forces method etc. [27]. In 1991, Evans et al. first used two micropipettes to unbinding two capsules with adhesion molecules [28]. Markel et al. measured the unbinding force between biotin and streptavidin by conjugated them to two capsules separately. With the method like Evans, capsules sucked by two micropipettes were moved away slowly
and the force was measured as capsules separated. [27][29]. The biotin-streptavidin unbinding force was about 50 pN. For optical tweezers method, Nishizaka et al.
measured the unbinding force between actin filaments in 1995 and the unbinding force was 9.2 ± 4.4 pN [30]. In 1996, Miyata et al. reported that the unbinding force measured by optical tweezers between actin and skeletal muscle α-actin was about 18 pN [31].
With the development of biotechnology, the interactions between different kinds of ligands and receptors become more and more important. A lot of different methods to investigate this kind of molecular interactions have been developed. In this thesis, we will focused on hydrodynamic shear assay and measure the aptamer-cTnI binding force by this method.