Convex lens-induced nanoscale templating SEE COMMENTARY Daniel J. Berarda, François Michauda, Sara Mahshida,b, Mohammed Jalal Ahameda, Christopher M. J. McFaula, Jason S. Leitha, Pierre Bérubéb, Rob Sladekb, Walter Reisnera,1, and Sabrina R. Lesliea,1 aDepartment of Physics, McGill University, Montreal, QC, Canada H3A 2T8; and bDepartment of Human Genetics, McGill University, Montreal, Canada H3A 0G1 Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved July 15, 2014 (received for review December 5, 2013) We demonstrate a new platform, convex lens-induced nanoscale These challenges have limited the practical range of nanochannel templating (CLINT), for dynamic manipulation and trapping of sin- dimensions to 40–50 nm (1). Moreover, the high hydraulic re- gle DNA molecules. In the CLINT technique, the curved surface of sistance of nanoscale features (for a slit of depth h, the hydraulic a convex lens is used to deform a flexible coverslip above a sub- resistance scales as 1/h3, compared with 1/h for electrical re- strate containing embedded nanotopography, creating a nano- sistance) (5) requires that electrophoretic actuation be used to scale gap that can be adjusted during an experiment to confine drive DNA into sub-100-nm nanochannels. This in turn neces- molecules within the embedded nanostructures. Critically, CLINT sitates the use of special high-salt electrophoresis buffers [2–5× has the capability of transforming a macroscale flow cell into a Tris/borate/EDTA (TBE)], which reduces DNA extension (6) and nanofluidic device without the need for permanent direct bond- constrains the imaging buffer used. One answer to these challenges ing, thus simplifying sample loading, providing greater accessibil- is to develop specialized grayscale lithography approaches (7, 8) ity of the surface for functionalization, and enabling dynamic that can create gently funneling channel dimensions, reducing the manipulation of confinement during device operation. Moreover, free-energy barrier. Gray-scale approaches, although they are as DNA molecules present in the gap are driven into the embedded feasible technologically, are still highly challenging to implement topography from above, CLINT eliminates the need for the high and remain limited in the range of confinement that can be varied pressures or electric fields required to load DNA into direct-bonded continuously in both lateral and vertical dimensions. nanofluidic devices. To demonstrate the versatility of CLINT, we To overcome the challenges faced by classical nanofluidic confine DNA to nanogroove and nanopit structures, demonstrating technology, we have developed a new approach for introducing DNA nanochannel-based stretching, denaturation mapping, and tunable nanoscale confinement to trap and align DNA molecules partitioning/trapping of single molecules in multiple embedded cav- for optical analysis. Our confinement-based imaging technology ities. In particular, using ionic strengths that are in line with typical combines nanotemplated substrates with a single-molecule im- biological buffers, we have successfully extended DNA in sub–30- aging technique called convex lens-induced confinement (CLIC) nm nanochannels, achieving high stretching (90%) that is in good (9). Fig. 1 illustrates a flow cell implementation of CLIC mi- agreement with Odijk deflection theory, and we have mapped ge- croscopy, in which molecules are initially loaded into a planar nomic features using denaturation analysis. micron-scale chamber (10). To form the CLIC imaging chamber, the upper chamber surface is subsequently pressed into contact single-molecule manipulation | polymer confinement | genomic mapping | with the lower surface using the curved surface of a lens (Figs. 1B CLIC imaging | nanotechnology and 2). The final vertical confinement profile varies gradually away from the contact point, typically increasing by tens of anoconfinement-based manipulation is a powerful approach nanometers over a field of view of a hundred microns. When Nfor controlling the conformation of single DNA molecules CLIC is performed over a surface containing nanotemplated on chip. When single polymer chains are squeezed into envi- ronments confined at length scales below their diameter of gy- Significance ration in free solution, the polymer equilibrium conformation will be molded by the surrounding nanoscale geometry. Nano- Convex lens-induced nanoscale templating (CLINT) represents channel arrays can be used for massively parallel extension of a conceptual breakthrough in nanofluidic technology for sin- DNA across an optical field, serving as the basis for a high- gle-molecule manipulation. CLINT solves a key challenge faced throughput optical mapping of genomes (1, 2). More varied by the nanofluidics field by bridging the multiple-length scales manipulations can be performed based on the design of the sur- required to efficiently bring single-molecule analytes from the rounding nanotopology, such as using nanocavities embedded in a pipette tip to the nanofluidic channel. To do this, CLINT loads nanoslit to trap single DNA molecules (3). Nanoconfinement- single-molecule analytes into embedded nanofeatures via dy- based manipulation, compared with competing techniques for namic control of applied vertical confinement, which we have single-molecule manipulation such as tweezer technology and demonstrated by loading and extending DNA within nano- surface/hydrodynamic-based stretching, has three key advantages channels. CLINT offers unique advantages in single-molecule DNA (4): (i) It is highly parallel, providing the high throughput essential mapping by facilitating surface passivation, increasing loading for mapping gigabase-scale mammalian genomes (1); (ii)itcanbe efficiency, obviating the need for applied pressure or electric efficiently integrated with microfluidics to rapidly cycle molecules fields, and enhancing compatibility with physiological buffers through the channel arrays for upstream/downstream pre- and and long DNA molecules extracted from complex genomes. postprocessing of DNA; and (iii) it does not require applied flow PHYSICS or electric force to maintain the DNA extension. Author contributions: W.R. and S.R.L. designed research; D.J.B., C.M.J.M., J.S.L., and S.R.L. performed research; D.J.B., F.M., S.M., M.J.A., C.M.J.M., J.S.L., P.B., R.S., W.R., and S.R.L. Nanoconfinement-based approaches have, however, a key dif- contributed new reagents/analytic tools; D.J.B., F.M., C.M.J.M., and S.R.L. analyzed data; ficulty inherent to the use of nanoscale dimensions: the need to and D.J.B., F.M., R.S., W.R., and S.R.L. wrote the paper. bridge length scales differing by up to 5 orders of magnitude The authors declare no conflict of interest. – (submillimeter scale of a pipette tip to channels in the 10 100 nm This article is a PNAS Direct Submission. range) in the same fluidic device. This introduces two challenges in See Commentary on page 13249. device design and manufacture: (i) the need to drive single-mol- 1To whom correspondence may be addressed. Email: [email protected] or reisner@ ecule analytes across a very high free-energy barrier at the edge of physics.mcgill.ca. nanoconfined regions and (ii) inefficient fluid transport due to the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BIOPHYSICS AND high hydraulic resistance of channels with nanoscale dimensions. 1073/pnas.1321089111/-/DCSupplemental. COMPUTATIONAL BIOLOGY www.pnas.org/cgi/doi/10.1073/pnas.1321089111 PNAS | September 16, 2014 | vol. 111 | no. 37 | 13295–13300 Downloaded by guest on September 25, 2021 AB volume interactions. If the width D of a square cross-section nanochannel is larger than the 50 nm persistence length P of the DNA, the polymer is described by the de Gennes confinement regime and will coil up into multiple blobs along the nano- 10 m 50nm channel axis (4). When D < P, coiling within the channel is suppressed and the molecule undergoes periodic deflections D L g along the walls with no back-bending (Odijk regime) (12). Fig. 1. Illustration of the DNA-loading procedure. (A) When the chamber height has microscale vertical dimensions, DNA molecules are unconfined A and take on coiled conformations. (B) When the push-lens is lowered, the imposed vertical nanoscale confinement causes DNA molecules to align in the Z-axis Piezo nanochannels, their energetically preferred state. Actuator Lens Rotation Mechanism structures such as nanocavities and nanogrooves, the vertical Z-axis Coarse Adjuster confinement imposed by CLIC drives the single-molecule ana- XY Translation “ lytes into the embedded topology. We call this approach convex Stage lens-induced nanoscale templating” (CLINT). Note that in CLINT, the molecules are loaded gently by imposing confine- ment from above, eliminating the need for high pressures or electric fields to introduce single-molecule analytes into the Push-Lens confined region of the device. This ease of loading ensures that Flow Cell the CLINT approach, when used to confine macromolecules in Microscope structures with dimensions smaller than their persistence length, Objective is compatible with a much wider range of ionic conditions than BC classic direct-bonded devices that rely on electrophoresis for loading. Loading molecules from above can also reduce the 50 3 mm 40 sensitivity of the technique to fabrication defects that can lead to 30 clogging of direct-bonded channels loaded from the side. We (nm) 20 demonstrate that CLINT can efficiently