NANOSLINKY: DNA ENTROPOPHORESIS DOWN A NANOFLUIDIC STAIRCASE E.A. Strychalski*, S.M. Stavis, M. Gaitan, and L.E. Locascio National Institute of Standards and Technology, USA

ABSTRACT Almost all nanofluidic devices for biopolymer analysis have been limited by one or two confining structural dimen- sions or the application of external forces for biopolymer manipulation, which has restricted the scope of related lab-on-a- chip technology. Here, a nanofluidic channel with a three dimensional (3D) “staircase” depth profile across its width was used to control and measure the behavior of individual DNA , which “descended” the confining staircase via “entropophoresis” effected by interactions between the DNA molecules and the nanofluidic step edges. Thus, the nanofabricated staircase enabled complex structural control over DNA transport and conformation without the applica- tion of external forces.

KEYWORDS: Nanofluidics, 3D, DNA, Entropophoresis, Separation,

INTRODUCTION Adaptation of the fabrication tools and techniques of the semiconductor electronics industry has fostered the develop- ment of miniaturized fluidic devices with engineered, nanometer-scale critical dimensions. Just as microfluidic structures have enabled new analytical techniques for biological entities with micrometer-scale characteristic dimensions, such as cells, nanofluidic structures are facilitating interrogation of nanoscale biological analytes, such as individual DNA molecules. In a nanofluidic device, a greater number of unique confining nanofluidic dimensions generally translates into more sophisticated analyte manipulation and characterization, but nearly all nanofluidic devices for biopolymer analysis have been limited by geometries consisting of one or two confining structural dimensions (with several notable excep- tions [1-4]). The requirement of external forces to transport DNA molecules, for example using hydrostatic or electroki- netic drive, has further restricted device design and utility. Here, a nanofluidic channel with a 3D “staircase” depth pro- file was used to control and measure the behavior of individual DNA molecules, which “descended” the confining stair- case by “entropophoresis” (Figure 1). Entropophoresis describes transport resulting from an entropy gradient and, in this work, denotes the Brownian motion of DNA molecules ratcheted by entropic forces applied discretely at nanofluidic step edges. In this way, the nanofabricated staircase enabled complex structural control over DNA transport and conformation without the application of external forces. The process of a DNA descending a nanofluidic staircase can be likened to what is arguably one of the most entertaining uses of a popular children’s toy in the United States of America called “Slinky”. A Slinky consists of a long, floppy, spring that is compact in its resting state. The Slinky can be placed at the top of a staircase and, as an initial con- dition, one end of the Slinky is pulled over the edge of the top step. This results in the spring “walking” itself end-over- end down the steps, alternately stretching between two steps and contracting while on a single step. This amusing event may assist in visualizing the experiment described here, wherein each DNA molecule placed at the “top” of a confining nanofluidic staircase was transported “down” the staircase via entropophoresis, much like a Slinky walking down a macroscopic staircase due to its momentum in the presence of a gravitational field. The DNA molecules also changed conformation, becoming more extended between steps and more compact while on an individual step. To complete the analogy, DNA molecules of different lengths, stiffness, or morphology exhibited different behaviors as they descended the nanofluidic staircase, just as Slinky toys of various sizes and stiffness walk down a macroscopic staircase differently.

EXPERIMENTAL A 3D nanofluidic device was fabricated as described previously in fused silica using grayscale photolithography, anisotropic reactive ion etching, and direct glass bonding [1]. The staircase consisted of 30 nanofluidic steps with widths of 4 µm, an average step height of 11 nm, and depths ranging from (1 ± 5) nm to (330 ± 4) nm (mean ± standard devia- tion). Figure 1A shows an optical micrograph of the bonded, air-filled device. The various colors arise from white light interference and indicate the staircase function depth profile, which is also depicted in Figure 1B in a scanning probe measurement across the channel width prior to bonding. Figures 1A and 1B are aligned to facilitate visualization of the 3D channel geometry. Four double-stranded DNA samples (Figure 2) were chosen to investigate behavior as a function of length, stiffness, and morphology. Linear DNA molecules (Bacteriophage-λ, 48.5 kilobasepair (kbp)) and circular DNA molecules (9-42 charomid, 42.2 kbp) were labeled with YOYO-1 at basepair:dye ratios of 5:1 and 20:1 to vary DNA stiffness. Molecules were driven electrokinetically “up” the staircase, the applied electric field was removed, and epifluorescence videomi- croscopy was used to observe individual DNA molecules at 5 s intervals (Figure 1A). The staircase was held at (27.0 ± 0.1) ºC and filled with 5X Tris Borate EDTA with 3 % β-mercaptoethanol. For each sample, 59 to 128 molecules were analyzed using custom MATLAB software.

U.S. Government work not protected by U.S. copyright 2071 14th International Conference on Miniaturized Systems for Chemistry and Sciences 3 - 7 October 2010, Groningen, The Netherlands Figure 1: Nanofluidic staircase and entropophoresis. (A) Optical micrograph of the 3D fused silica nanofluidic channel. The staircase function depth profile is made visible by white light interference patterns. Structural control of DNA transport down the staircase by entropophoresis is illustrated (DNA not to scale). (B) Scanned probe measurement of the nanofluidic channel depth across the channel width. The numerous depths of the nanofluidic channel form a com- plex entropic free energy landscape to manipulate and study the behavior of single DNA molecules.

Figure 2: Single molecule fluorescence microscopy. Unprocessed epifluorescence micrographs show individual DNA molecules labeled with YOYO-1 assuming larger molecular conformations in the shallower (A-D) than the deeper (E-H) regions of the nanofluidic staircase, due to the confining geometry. DNA samples are: (A, E) linear 5:1; (B, F) linear 20:1; (C, G) circular 5:1; and (D, H) circular 20:1.

RESULTS AND DISCUSSION The staircase geometry is analogous to a collection of nanoslits of increasing depth arrayed across the channel width and, as such, constitutes a natural extension of previous studies examining DNA behavior in confining nanoslits, each with a single depth (for example, references [2, 5]). A distinguishing difference of the current study is that the same DNA molecule was observed in dynamic transitions between differing degrees of confinement, in addition to confinement at a single device depth. The various DNA samples generally displayed similar qualitative behavior: molecules proceeded down the staircase by entropophoresis; molecular conformation depended on step depth (Figure 2); and, molecules were transported through at least two regimes of confinement [2, 5]. Quantitative differences in behavior could be exploited for concentrating that collect at the bottom of the staircase or separating biopolymers with different mor- phologies and stiffness, such as double-stranded DNA, single-stranded DNA, RNA, or .

2072 CONCLUSION The measurements described here constitute a comprehensive examination of confined DNA behavior in unprecedent- ed detail and represent a fundamental advance in experimental polymer studies needed to progress nanofluidic lab-on-a- chip technology. Although the nanofluidic staircase is conceptually simple and relatively straightforward to fabricate, the geometry is complex, enabling new measurement capabilities as individual biomolecules dynamically navigate the resul- tant complex free energy landscape. Such devices are fully compatible with optical microscopy and real-time single molecule analysis techniques and could be multiplexed and integrated into a lab-on-a-chip analytical platform. These re- sults suggest a new paradigm for nanofluidic functionality and metrology with applications relevant to lab-on-a-chip tech- nology, such as sample preparation (for example, separation and concentration), fundamental polymer studies (including nanoscale confinement and transport of biopolymers), and single molecule manipulation (such as directed self-assembly of molecular constructs).

ACKNOWLEDGEMENTS This work was performed in part while E.A.S. and S.M.S. held National Research Council Research Associateship Awards. Device fabrication was performed at the Cornell Nanoscale Science and Technology Facility (CNF), a member of the National Nanotechnology Infrastructure Network, and the Cornell Center for Materials Research, both supported by the National Science Foundation. Device characterization was performed in part at the National Institute of Standards and Technology Center for Nanoscale Science and Technology. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States. Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification implies neither recommendation or endorsement by the National Institute of Standards and Technology nor that the materials or equipment identified are necessarily the best available for the purpose.

REFERENCES [1] S.M. Stavis, E.A. Strychalski, and M. Gaitan, Nanofluidic structures with complex three-dimensional surfaces, Nan- otechnology, vol. 20, 165302, (2009). [2] E.A. Strychalski, S.M. Stavis, and H.G. Craighead, Non-planar nanofluidic devices for single molecules analysis fabricated using nanoglassblowing, Nanotechnology, vol. 19, 315301 (2008). [3] H. Cao, J.O. Tegenfeldt, R.H. Austin, and S.Y. Chou, Gradient Nanostructures for Interfacing Microfluidics and Nanofluidics, Applied Physics Letters, vol. 81, 3058, (2002). [4] S.M. Stavis, J. Geist, M. Gaitan, Separation and Metrology of by Nanofluidic Size Exclusion, Lab on a Chip, in press, (2010). [5] D. J. Bonthuis, C. Meyer, D. Stein, C. Dekker, Conformation and Dynamics of DNA confined in Slitlike Nanoflu- idic Channels, Physical Review Letters, vol. 101, 108303 (2008).

CONTACT *E.A. Strychalski, tel: +1-301-975-5951; [email protected]

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