6.3 Results and Discussion
6.3.1 Strength of attachment
The end-to-end distance of bound DNA has been described as a suitable parameter to estimate the molecules’ binding strength.7 DNA molecules strongly bound to a surface will resemble rigid projections of their 3-D confirmations upon adsorbing to the surface, whereas loosely bound molecules will be able to equilibrate to a 2-D conformation.7,35 Surface irregularities and DNA overlapping / aggregation observed at 1 and 5mM salt concentrations hindered effective calculation of end-to-end distances; but, in general, molecules bound to BGR surfaces appeared to become more condensed with increased cation concentration, suggesting that additions in divalent salt promote stronger binding of DNA (Figure 6.7-6.10).
Images collected from solutions containing ZnCl2, possessed the largest amount of dried salt as compared to the other cation solutions (Figure 6.7a-c). DNA molecule conformations were highly condensed when in contact with salt precipitates (Figure 6.7c). Molecules bound along {210}r intergrowth boundaries in the presence of 1 and 5 mM and ZnCl2 possess much smaller end-to-end distances (in most cases, the molecules were too entangled to measure effective distances) than those bound to rutile-rich surface regions (Figure 6.7b,c) .
The DNA molecules bound in the presence of 0.5 mM NiCl2 appeared to be aligned in a given direction (believed to be the direction of the receding meniscus 36,37).
At this salt concentration the majority of DNA molecules appear to attach at their endpoints, with the remainder of the molecule being pulled in the direction of the receding meniscus (Figure 6.8a). As the salt concentration of the solution was increased to 1mM, the molecules bound to the BGR surface appeared to become more collapsed and condensed, suggesting a stronger binding to the surface (Figure 6.8b). At 5 mM, an increase in dried salt (globular shaped) is observed lying predominately along {210}r
Figure 6.8. AFM images (1.5 x 1.5 µm) of BGR surfaces exposed to DNA solutions containing additions of: a) 0.5 mM NiCl2 (z-scale: 15nm). The white arrow describes the assumed direction of the receding meniscus. The associated force of the meniscus is believed to explain the aligned orientation of bound molecules. b) 1.0 mM NiCl2
(phase image, z-scale: 20o) c) 5.0 mM NiCl2 (z-scale: 15nm). As the concentration of NiCl2 is increased the conformation of bound DNA molecules appears to become more condensed, and an increase in salt precipitates is observed.
b)
c) a)
Figure 6.9. AFM images (1.5 x 1.5 µm, z-scale: 15nm) of BGR surfaces exposed to DNA solutions containing additions of: a) 0.5 mM CoCl2. b) 1.0 mM CoCl2. c) 5.0 mM CoCl2. As the concentration of CoCl2 is increased the conformation of bound DNA molecules appears to become more condensed.
a)
c)
b)
Figure 6.10. AFM images (1.5 x 1.5 µm) of BGR surfaces exposed to DNA solutions containing additions of: a) 0.5 mM MgCl2 (z-scale: 15nm). b) 1.0mM MgCl2 (z-scale:
15nm). c) 5.0 mM MgCl2 (phase image, z-scale: 20o). d) 5.0 mM MgCl2 (phase image, z-scale: 20o). Images c and d in the presence of 5 mM MgCl2 exhibit a high degree of preferential attachment of DNA along {210}r intergrowth boundaries.
a) b)
c) d)
intergrowth boundaries (Figure 6.8c). Observing a greater degree of salt deposition along {210}r intergrowth boundaries supports the belief that the divalent cations are
preferentially attaching to tunnel sites within these linear boundaries, subsequently facilitating the attachment of DNA.
Solutions containing Co (II) additions appeared to follow similar trends observed within the other cation-containing solutions (Figure 6.9a-c). Molecules bound to {210}r
boundaries appeared to become more condensed with increases in cation concentration (Figure 6.9a-c). At a concentration of 0.5 mM CoCl2 (Figure 6.9a) the DNA molecules did not appear to be as extended as they were while in the presence of 0.5mM NiCl2
(Figure 6.8a), suggesting that at this level of salt concentration, Co (II) cations bind DNA molecules more tightly than Ni (II) cations. At the 5.0 mM concentration, there did not appear to be as many salt deposits as those observed in the presence of NiCl2 or ZnCl2, so highly condensed DNA conformations were not as prevalent.
Solutions containing 0.5 and 1.0 mM MgCl2 behaved similarly to other cation- containing solutions (Figure 6.10a,b); a decrease in end-to-end distance was observed while increasing MgCl2 concentration. The binding of DNA molecules along {210}
boundaries in the presence of 5.0 mM MgCl2 differed from the adsorption behavior observed with the other divalent cations tested. At 5.0 mM MgCl2, both highly condensed, and outstretched molecules were observed (Figure 6.10c). The outstretched behavior observed within the Mg (II) samples is not believed to be due to a receding meniscus force (as observed at 0.5 mM NiCl2 solutions) since the orientation of bound molecules is not uniform in one direction (compare Figure 6.10a to Figure 6.8a), but rather aligned along {210}r intergrowth boundaries. This difference in binding behavior could be based in the interaction between the divalent cation and the DNA molecule.
According to Izatt et al., the Mg (II) cation binds primarily to the phosphate groups along the DNA backbone; whereas the affinity for nucleotide base-binding in relation to phosphate binding increases in the following order: Mg (II), Co (II), Ni (II), Mn (II), Zn (II), Cd (II), and Cu (II).38,39 In DNA solutions containing Co (II), Ni (II), and Zn (II) additions, a higher degree of base binding could lead to higher affinities for molecular aggregation from base – base hybridization;29 whereas the Mg (II) cations’ higher affinity for phosphate binding sites could lead to a reduction in molecular aggregation.