Localized nanoscopic surface measurements of nickel-modified mica for single-molecule DNA sequence sampling.

Engineered planar substrates, such as chemically-derivatized glass, silicon/silicon oxide, and gold-thiol-based self assembled monolayers (SAMs) are critical enabling components in widely used and commercially important technologies such as ‘next gen’ DNA sequencing9, 10, nucleic acid microarrays11, and surface plasmon resonance biosensors (Biacore, etc)12. Because most of these technologies employ far-field optical microscopy or photonic detection, surface topography and nanometer-scale variations in surface chemistry of the substrate is not of concern. However, in scanning probe microscopy of nucleic acids and nucleic acid-protein complexes, substrate surface topography and local physico-chemical properties are critical issues. When used with the proper attention to substrate:sample preparation, scanning probe microscopy offers substantial advantages over photonic detection in terms of sensitivity (no labels), resolution (sub-molecular-to-atomic) and breadth of measurement modalities (topography, stiffness, adhesiveness, as well as thermal and electrostatic properties). Paradoxically, substrates used in scanning probe microscopy tend to be less engineered and less well characterized than are scaffolds and surfaces used in systems with photonic detection. This is particularly true in the of study of biopolymers, such as DNA and protein fibers of various kinds, which can be microns-to-millimeters in length and thus require vast areas of atomically-flat surface that can be very difficult to achieve on chemically modified glass or gold:thiol:SAM substrates. Freshly cleaved mica complexed with multi-valent cations is a very popular but simple system used to bind DNA for AFM imaging in liquid and in air. In particular, Nickel (II) ion, applied in the form of an aqueous chloride or nitrate salt solution, has been shown to produce unusually strong DNA adsorption on mica compared to similar divalent metal ions and this has been used to advantage in a number of studies, including our own13, where strong adsorption is required1, 4. Despite Ni:mica’s widespread use by the AFM community and its potential as a substrate for DNA nanotechnologies of various kinds, knowledge of its nanoscale physico-chemistry is limited. A series of classic studies used surface force apparatus (SFA) to study the interaction between two cleaved mica surfaces in various mono- and divalent aqueous metal-ion electrolytes14, 15 (though not including Ni2+). These authors analyzed the mica-mica interaction in terms of an extended DLVO theory of colloid stability. Notably, their experiments were mostly conducted at pH 5.8. Workers studying the biochemistry of single DNA molecules bound to mica with AFM have extended this classic SFA-based work, using a generalization of the DLVO theory to model the force interaction of a charged DNA molecule and mica surface, rather than two mica surfaces, in aqueous metal ion electrolytes at physiological pH (7.0–8.5)4, 5, 7, 8. However, metal ions in solution are known to exhibit complex speciation behavior16, and this important aspect of metal ion chemistry has not been taken into account in the past modeling and discussion of DNA binding to mica. Further, the nickel-ion treated mica surface itself has not been directly characterized due to the minute quantities of material involved. In this report, we use a combination of nanoscale analytical techniques to probe the properties of Ni:mica on the molecular scale. We employ an exquisitely sensitive surface analysis technique, secondary ion mass spectrometry to confirm in situ partial ion exchange between Ni2+ in the solution and native K+ present on the untreated mica surface. Using AFM we show that a smooth, stable but difficult-to-detect molecular monolayer can form on mica treated with aqueous NiCl2 at pH ~7, likely a result of metal or metal-hydroxide precipitates. Importantly, this result indicates that the actual substrate:molecule interaction can be mediated in some cases by a molecular adlayer of different physico-chemical properties than the mica itself. We present thermodynamic simulations which show that nickel-ion speciation in aqueous solutions is highly sensitive to pH in the range 6–8. As a consequence the solution concentration of the divalent species can vary widely from what is assumed, and the speciation of other metal ions-complexes can be unpredictable unless great care is taken to stabilize the pH condition and redox condition of the solution. Taken together, our simulation and experimental results strongly suggest more detailed modeling and experiments are needed to properly understand and control the mica:metal-ion:DNA system at physiological pH. In order to determine the biofunctional characteristics of the Ni:mica surfaces we digested mica-adsorbed plasmid DNA with a restriction enzyme, RsaI. We show that Ni:mica is compatible with enzymology conducted directly on the surface-fixed DNA molecules, and can be used to precisely locate DNA sequence motifs within single molecules17. Similarly, it has potential uses as a scaffold or substrate for engineering DNA nanostructures and in other areas of DNA nanotechnology, like single molecule DNA sequencing.