To explore the analytic relevance of unbinding force measurements between complementary DNA strands with an atomic force microscope, we measured the forces to mechanically separate a single DNA duplex under physiological conditions by pulling at the opposite 5-ends as a function of the loading rate (dynamic force spectroscopy). We investigated DNA duplexes with 10, 20, and 30 base pairs with loading rates in the range of 16-4,000 pN͞s. Depending on the loading rate and sequence length, the unbinding forces of single duplexes varied from 20 to 50 pN. These unbinding forces are found to scale with the logarithm of the loading rate, which is interpreted in terms of a single energy barrier along the mechanical separation path. The parameters describing the energy landscape, i.e., the distance of the energy barrier to the minimum energy along the separation path and the logarithm of the thermal dissociation rate, are found to be proportional to the number of base pairs of the DNA duplex. These single molecule results allow a quantitative comparison with data from thermodynamic ensemble measurements and a discussion of the analytic applications of unbinding force measurements for DNA.The direct measurement of forces between individual biological ligand receptor pairs is an emerging field. With the atomic force microscope (AFM), it has been possible to measure unbinding forces between single ligand receptor pairs, which are typically in the pico-Newton range (1-5). These sensitive force measurements can be performed with a high spatial resolution (5). However, the relevance of the force measurements as an (local) analytical tool depends on the understanding of the relationship between the measured forces and thermodynamic data characterizing the complex (6).The measured unbinding forces are not a fundamental property of a ligand-receptor pair but depend on the loading rate that is applied to the complex: If the load on the complex increases sufficiently slowly, there is time for thermal fluctuations to drive the system over the energy barrier, and the unbinding force will be small (7). A scaling of the force with the logarithm of the loading rate is expected for a single energy barrier along the unbinding path (8) and was found in AFM experiments on unfolding of protein domains (9, 10) and on rupture force measurements of the P-selectin͞ligand complex (11). With a different force probe technique, it has been possible to measure the loading rate dependence of the unbinding forces of biotin͞(strept)avidin complexes (12). In this system the loading rate dependence of the force is more complicated, indicating that more than one energy barrier is present along the separation path.The aim of our paper is to present a deeper understanding of the forces that arise if one separates the two strands of a DNA duplex by pulling at both 5Ј-ends. For this purpose, we have measured the loading rate dependence of the unbinding forces between complementary DNA strands to get information about the energy profile of the separation...

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3 and 5 overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.

Their analytic approximation for the elasticity is given by Fp/ k b T ϭ 0.25*(1Ϫ x/L) Ϫ2 Ϫ 0.25 ϩ x/ L, where F is the force applied across the ends, p is the persistence length, and k B T is the thermal energy.