Chemical force microscopy (CFM) was used to probe interactions between ionizable and neutral functional groups in aqueous solutions. Force microscope probe tips and sample substrates have been covalently modified with self-assembled monolayers (SAMs) terminating in distinct functional groups. SAMs were prepared by treating Au-coated or uncoated tips and substrates with functionalized thiols or silanes, respectively. A force microscope has been used to characterize adhesive and frictional interactions between probe tips and substrates modified with SAMs that terminate in -NH 2 , -COOH, -OH, and -CH 3 functional groups as a function of solution pH and ionic strength. In general, adhesion and friction forces were observed to depend on the chemical identity of the tip and sample surface and also were found to be highly sensitive to the changes in the ionization state of the terminal functionalities induced by varying the solution pH. Adhesion measurements made as a function of pH (force titrations) on amine-terminated surfaces exhibit a sharp decrease in the adhesion force at pH values below 4.5. The pK a estimated from this drop in adhesion, which is due to the protonation of amine groups on the sample and tip, 3.9, is similar to the value determined by conventional contact angle wetting studies of these same surfaces. The large decrease in the pK of the amine group relative to homogeneous solution was attributed to the relatively hydrophobic environment of the amine group in these SAMs. Adhesion measurements made as a function of pH on -COOH-terminated surfaces exhibited a large drop in adhesion force for pH values greater than 5. The pK a estimated from these data, 5.5, is similar to the free aqueous solution value. In addition, the adhesion force between nonionizable -OH and -CH 3 groups was found to be independent of solution pH. The measured adhesive forces were interpreted using a contact mechanics model that incorporated the effects of the double layer free energy. Analyses of repulsive electrostatic forces and adhesion data recorded as a function of ionic strength were used to determine properties of the double layer. The pH dependence of the friction force between tips and samples modified with SAMs terminating in -COOH, -OH, and -CH 3 groups was measured as a function of applied load. For a given pH, these data exhibit a linear dependence on load with the slope corresponding to the coefficient of friction. The coefficient of friction for -OH and -CH 3 groups was independent of pH, while the friction coefficient for -COOH-terminated surfaces drops significantly at a pH corresponding to the pK a determined by adhesion measurements. The pHdependent changes in friction forces for ionizable groups were exploited to map spatially changes in ionization state on surfaces terminating in -COOH and -OH functional groups.

Atomic force microscopy is an imaging tool used widely in fundamental research, although it has, like other scanned probe microscopies, provided only limited information about the chemical nature of systems studied. Modification of force microscope probe tips by covalent linking of organic monolayers that terminate in well-defined functional groups enables direct probing of molecular interactions and imaging with chemical sensitivity. This new chemical force microscopy technique has been used to probe adhesion and frictional forces between distinct chemical groups in organic and aqueous solvents. Contact mechanics provide a framework to model the adhesive forces and to estimate the number of interacting molecular groups. In general, measured adhesive and frictional forces follow trends expected from the strengths of the molecular interactions, although solvation also plays an important role. Knowledge of these forces provides a basis for rationally interpretable mapping of a variety of chemical functionalities and processes such as protonation and ionization.

We measured the force required to peel single-stranded DNA molecules from single-crystal graphite using chemical force microscopy. Force traces during retraction of a tip chemically modified with oligonucleotides displayed characteristic plateaus with abrupt force jumps, which we interpreted as a steady state peeling process punctuated by complete detachment of one or more molecules. We were able to differentiate between bases in pyrimidine homopolymers -peeling forces were 85.3 ±4.7 pN for polythymine and 60.8±5.5 pN for polycytosine, substantially independent of salt concentration and the rate of detachment. We developed a model for peeling a freely jointed chain from the graphite surface and estimated the average binding energy per monomer to be 11.5±0.6 k B T and 8.3±0.7 k B T in the cases of thymine and cytosine nucleotides. The equilibrium free-energy profile simulated using molecular dynamics had a potential well of 18.9 k B T for thymidine, showing that non-electrostatic interactions dominate the binding. The discrepancy between the experiment and theory indicates that not all bases are adsorbed on the surface or that there is a population of conformations in which they adsorb. Force spectroscopy using oligonucleotides covalently linked to AFM tips provides a flexible and unambiguous means to quantify the strength of interactions between DNA and a number of substrates, potentially including nanomaterials such as carbon nanotubes.Interactions of polyelectrolytes with solid substrates have several important applications in materials science and engineering. 1, 2 On complexation with neutral particles, polyelectrolytes convert these particles into charged species, enabling dispersions in aqueous media, and find use in detergents, cosmetics, gels, food additives, and oil recovery. In a biological context, our interest is in the interaction between single and double-stranded DNA (ssDNA and dsDNA) and substrates such as graphite and carbon-nanotubes (CNTs) that can potentially play a vital role in biomedicine, 3, 4 nanotechnology, 5,6 and relevant for the understanding of the origin of life. 7 Importantly, ssDNA has been used successfully for dispersion and solution-based manipulation of CNTs -ssDNA forms a stable hybrid with a nanotube by wrapping around the CNT in helical fashion. 8,9 The hybrid is useful for dispersion, sorting, 8,9 and patterned placement of nanotubes, 6 for transportation of DNA into cells, and for killing cancer cells by thermal ablation. 3 Strength of dispersion, ability to sort, and stability in the cellular environment all depend on the interaction between DNA and a CNT. 10 Individual DNA bases also bind to a graphitic surface through non-covalent π-π interactions. [11][12][13][14] Very little is known quantitatively about the strength of binding between ssDNA and CNT's in spite of the fundamental importance of understanding such interactions. 8,[15][16][17] As a first

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to $100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.