3.1 Effect of ss-DNA Concentrations on ss-DNA Adsorption on Mica Surfaces
To explore the lowest ss-DNA concentration that ss-DNA can form a thin-layer on mica surface, ss-DNA were incubated in 1 mmol/L CoCl2 solution with various ss-DNA concentrations. Figure 2(a) reveals the force-distance profiles measured with SFA, where open symbols for in-run and solid symbols for out-run. A slight steric repulsion emerges and then hinders surfaces from approaching when two mica are driven into contact. With further approach, the profiles appear a vertical area where force increases rapidly but distance nearly keeps constant even under load beyond 15 mN/m. In SFA experiment, such constant distance DH, typically defined as the hardwall distance, is used to characterize the thickness of adsorbed layer on the substrate, as illustrated in Figure 1(b).
In the case of 25 ng/μL, the average hardwall is 0.16±0.1 nm, meaning few or no ss-DNA binds to mica within measurement error; As DNA concentration increases to 50 ng/μL, the hardwall distance increases to 1.49±0.2 nm. Considering the measured hardwall distance is equivalent to the dynamic diameter of ss-DNA molecules of 1.2‒1.4 nm in solution [40], this hardwall presents a dense monolayer composed of ss-DNA molecules flat-lying on mica substrate, as illustrated in Figure 2(b). This is greatly consistent with the previous studies, revealing the formation of close-packed DNA monolayer on mica surface in CoCl2 solution containing various DNA (λ-DNA [12, 13], plasmid DNA [12, 35] or linear DNA [12]). As DNA concentration further increases to 100 ng/μL, a larger hardwall distance of 2.13±0.2 nm implies an ordered ss-DNA bilayer or a cross-link configuration that DNA molecules cross or overlap each other, which is shown in Figure 2(c). The expected cross-link or condensation configuration of DNA on mica has been reported previously [12, 41], which further identify the above explanation about ss-DNA geometry for DH = 2.13 nm. These adsorptions are tight and stable enough due to the reproducible measurements even beyond 15 mN/m.
In order to further demonstrate ss-DNA did adsorb on mica substrate, the adsorption structure of 100 ng/μL ss-DNA on mica surface was further characterized with AFM, shown in Figure 3. The sample preparation is the same as before, except the sample was dried with N2 at last. It can be found that almost the whole mica surface is covered with condensed wormlike ss-DNA molecules. The surface roughness of adsorbed ss-DNA film is ~1.8±0.2 nm in Figure 3(a), which was thought to be the relative height of adsorbed film from the mica surface, approximately corresponding to the hardwall distance measured in the previous SFA experiments.
Furthermore, Figure 3(c) reveals the roughness of mica surface is less than 0.15 nm, which is smooth enough to be used as a substrate in DNA absorption investigations. However, after ss-DNA absorption illustrated in Figure 3(b), the mica surface becomes uneven and has a high and uniform surface coverage of ss-DNA. To sum up, these AFM results certifies the adsorption morphology and configuration of ss-DNA measured by SFA.
In addition to the difference in configuration, the nano-mechanical properties are also presented in Figure 2(a). An adhesive force between DNA-coated mica and bare mica were measured during surface separation. “Jump in” phenomenon was found only at DNA concentration less than 50 ng/μL and “jump out” in all DNA conditions (note: jump in and jump out phenomenon occur in the case of the interaction force larger than cantilever-spring force when surface approach and separation respectively). Under the condition of less than 25 ng/μL (data of 10 ng/μL not shown), the adhesion reaches −37.5±0.2 mN/m, which is six times higher than those at 50 and 100 ng/μL. However, this value is roughly consistent with the force between two bare micas in the same solution (as discussed below), which further proved few or no ss-DNA absorb on mica at 25 ng/μL. Furthermore, the adhesive force decreases to −6.5±0.2 mN/m (Ead≈−1.38 mJ/m2) at 50 ng/μL and −5.3±0.2 mN/m (Ead≈−1.12 mJ/m2) at 100 ng/μL, which are slightly higher than previous studies [13], perhaps due to different CoCl2 concentration. These adhesive forces far lower than those between two bare micas actually prove ss-DNA molecules have been assuredly immobilized on mica. Additionally, the adhesive force at 100 ng/μL is slightly less than 50 ng/μL, which was due to the stronger steric effect induced by the more adsorbed ss-DNA.
Generally, the counterion-correlation effect is believed to be the dominant cause of dsDNA adsorption on mica mediated by divalent cations [12, 33]. Therefore, it is also expected that ss-DNA molecules are capable to adsorb onto mica surface immersed in divalent cation solution with the sharing of the ss-DNA and mica counterions. In this case, divalent salt acts as bridge between ss-DNA and mica, thus producing a net attraction that pulls ss-DNA onto mica surface. However, the measured force is greater than that of others who only ascribed adsorption to ion-correlation [33]. This is due to the cooperative effect of divalent metal ions condensation along DNA and their reaction with the surface groups [32]. The electrostatic potentials of DNA induce the increase of surface divalent ions concentration, which promotes the reaction of divalent ions with the surface and further results in the stronger ss-DNA adsorption.
3.2 Desorption of ss-DNA from Mica Surface with Excessive Monovalent Counterion
It is generally believed the competition effect between monovalent and divalent salts in solutions may result in the release of dsDNA molecules from mica surface [27, 33, 35]. In this study, the desorption behavior of absorbed ss-DNA layer from mica was observed by introducing monovalent salts into the gap buffer. As shown in Figure 4, once ss-DNA adsorption in 1 mmol/L CoCl2 (marked as Step 1), the gap buffer was completely replaced with excessive 1 mmol/L NaCl (Step 2), finally the gap buffer was returned back to 1 mmol/L CoCl2 (Step 3). The interaction between two surfaces in Step 1 is the same as that of Figure 2 at 50 ng/μL: the hardwall (~1.48 nm) was equal to a monolayer as before shown in Figure 4(b).
It is intriguing to note that the measured hardwall changes dramatically in different Steps. The hardwall decreases sharply to ~0.34 nm once the gap buffer was replaced to NaCl (Step 2 in Figure 4). Considering the measurement error, the sharp decrease in hardwall reveals desorption of ss-DNA layer from mica does occur in 1 mmol/L NaCl. Additionally, a long-range repulsion at a distance of 25±2 nm in Step 2 is observed in Figure 4(a), which is likely ascribed to the cooperative combination of double-layer force from electrolyte solution and steric repulsion induced by the released ss-DNA suspended in buffer solution shown in Figure 4(c). In the last step, the hardwall increases to 0.84±0.2 nm, which is far less than that of Step 1. This none-zero hardwall may be due to that the residual ss-DNA in solutions immediately reabsorb onto mica surface once CoCl2 was added, as shown in Figure 4(d).
The desorption strength of ss-DNA is monitored by the binding competition between monovalent and divalent cations on mica surface. Based on Poisson-Boltzmann equation and site-binding model [42], if monovalent salts were added to the bulk solution containing divalent salts, the divalent and monovalent cations would compete fiercely with each other for the binding sites of mica surface and DNA, thus decreasing markedly the surface density of divalent cations, which directly weakens the binding strength of DNA molecules on mica surface [33]. Therefore, the loosely bound ss-DNA can be easily squeezed out in monovalent salts solution, thus resulting in the desorption of ss-DNA from mica surface.
In addition, the nano-mechanical properties were also measured for studying the desorption behavior of ss-DNA thoroughly. In Step 1, the adhesive force (−5±0.2 mN/m) is consisted with that in Figure 2 within error. However, after the gap buffer was replaced by monovalent salts and returned back to divalent salts, an interesting feature is found in Step 3: the adhesion decreases to half of the original magnitude when the buffer was returned back, which may be due to that the binding sites of mica and DNA sites are occupied by Na+ [33]. All in all, the above phenomenon implies the cobalt ions play a crucial role in binding energy between adsorbed monolayer and bare mica surface.
To further explore the desorption of ss-DNA, a control experiment between two bare mica surfaces was conducted in the same buffer solutions without ss-DNA. As shown in Figure 5, the hardwall is close to zero in both CoCl2 solution, but increases to 0.65 nm in NaCl solution, which is likely due to the hydration of Na+ [43]. The experimental process is the same as the Steps of Figure 4, except no ss-DNA in the buffer solutions. As discussed above, the much higher adhesion in Step 1 than that of Figure 4 verifies ss-DNA absorption does occur. Comparing the Step 3 with Step 1, the force-distance profiles are almost the same: no long-range repulsion is found and two surfaces jump into contact from ~5 nm due to van der Waals interaction. However, in Step 2, the long-range repulsion observed on approaching comes from the electrostatic double-layer force and the initial distance of repulsion: ~10.6 nm is roughly equivalent to the theoretical Debye length: 9.6 nm. In brief, this exploration further explains the effects of some factors (ion species, ion valence and DNA concentrations) on the ss-DNA desorption behaviors.
As we know, due to the competition effect, the decrease in surface concentration of divalent cations by adding monovalent salts will directly weaken the binding affinity of ss-DNA on mica substrate. Meanwhile, high surface density of monovalent cations also inhibits DNA from attracting to mica surface. Each of these is responsible for the reduction of ion-correlation effect, which is believed to be the dominant cause of ss-DNA adsorption. Therefore, the desorption of ss-DNA layer in monovalent salts solution is mainly ascribed to the reduced ion-ion correlation force due to the sharp reduction of Co2+ surface concentration. Furthermore, Na+ in buffer solution may occupy the binding sites on both mica surface and ss-DNA molecules, thus screening the binding sites for Co2+ binding to ss-DNA [35]. In this case, the loosely bound ss-DNA on mica surface can be easily squeezed out under high loads.