We demonstrate a new platform, convex lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a convex lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub-30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.single-molecule manipulation | polymer confinement | genomic mapping | CLIC imaging | nanotechnology N anoconfinement-based manipulation is a powerful approach for controlling the conformation of single DNA molecules on chip. When single polymer chains are squeezed into environments confined at length scales below their diameter of gyration in free solution, the polymer equilibrium conformation will be molded by the surrounding nanoscale geometry. Nanochannel arrays can be used for massively parallel extension of DNA across an optical field, serving as the basis for a highthroughput optical mapping of genomes (1, 2). More varied manipulations can be performed based on the design of the surrounding nanotopology, such as using nanocavities embedded in a nanoslit to trap single DNA molecules (3). Nanoconfinementbased manipulation, compared with competing techniques for single-molecule manipulation such as tweezer technology and surface/hydrodynamic-based stretching, has three key advantages (4): (i) It is highly parallel, providing the high throughput essential for mapping gigabase-scale mammalian genomes (1); (ii) it can be efficiently integrated with microfluidics to rapidly cycle molecules through the channel arrays for upstream/downstream pre-and postprocessing of DNA; and (iii) it does not require applied flow or electric force to maintain the DNA extension.Nanoconfinement-based approaches have, however, a key difficulty inherent to the use of nanoscale dimensions: the need to bridge length scales differing by up to 5 orders...

We directly measure the free energy of confinement for semiflexible polymers from the nanoscale to bulk regimes in slit-like confinement. We use convex lens-induced confinement (CLiC) microscopy of DNA to directly count molecules at equilibrium in a single chamber of smoothly increasing height. Our data, acquired across a continuum of confinement regimes, provide a bridge with which to connect scaling theories established for qualitatively different regimes. We present new experimental data and simulations that connect the Odijk theory describing sub-persistence-length confinement, the interpolation model by Chen and Sullivan extending Odijk to moderate confinement, and the Casassa theory describing the transition from moderate confinement to bulk. Further, this work establishes a robust, quantitative platform for understanding and manipulating biopolymers at the nanoscale, with key applications and insights toward emerging genomic analysis tools.

We present the conception, fabrication, and demonstration of a versatile, computer-controlled microscopy device which transforms a standard inverted fluorescence microscope into a precision single-molecule imaging station. The device uses the principle of convex lens-induced confinement [S. R. Leslie, A. P. Fields, and A. E. Cohen, Anal. Chem. 82, 6224 (2010)], which employs a tunable imaging chamber to enhance background rejection and extend diffusion-limited observation periods. Using nanopositioning stages, this device achieves repeatable and dynamic control over the geometry of the sample chamber on scales as small as the size of individual molecules, enabling regulation of their configurations and dynamics. Using microfluidics, this device enables serial insertion as well as sample recovery, facilitating temporally controlled, high-throughput measurements of multiple reagents. We report on the simulation and experimental characterization of this tunable chamber geometry, and its influence upon the diffusion and conformations of DNA molecules over extended observation periods. This new microscopy platform has the potential to capture, probe, and influence the configurations of single molecules, with dramatically improved imaging conditions in comparison to existing technologies. These capabilities are of immediate interest to a wide range of research and industry sectors in biotechnology, biophysics, materials, and chemistry.

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid’s structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

We demonstrate a lab-on-a-chip that combines micro/nano-fabricated features with a Convex Lens-Induced Confinement (CLIC) device for the in situ analysis of single cells. A complete cycle of single cell analysis was achieved that includes: cell trapping, cell isolation, lysis, protein digestion, genomic DNA extraction and on-chip genomic DNA linearization. The ability to dynamically alter the flow-cell dimensions using the CLIC method was coupled with a flow-control mechanism for achieving efficient cell trapping, buffer exchange, and loading of long DNA molecules into nanofluidic arrays. Finite element simulation of fluid flow gives rise to optimized design parameters for overcoming the high hydraulic resistance present in the micro/nano-confinement region. By tuning design parameters such as the pressure gradient and CLIC confinement, an efficient on-chip single cell analysis protocol can be obtained. We demonstrate that we can extract Mbp long genomic DNA molecules from a single human lybphoblastoid cell and stretch these molecules in the nanochannels for optical interrogation.