Biomolecular Applications of Entropic Traps

Joshua Cross, Kevan Samiee, Elizabeth Strychalski

We have made nanoscale constrictions in fluid channels of varying size and orientation as entropic traps to investigate the dynamics of individual DNA molecules as they probe and subsequently escape through the trap. Previous researchers have made or modeled entropic traps in multiple configurations for separating DNA molecules or for studying the motion of DNA molecules through entropically restrictive spaces [1-3,5]. This work has focused on obtaining the time that individual molecules spend at the boundary between entropically favorable and unfavorable regions when driven electrophoretically.

We fabricated entropic traps of various size and orientation in fused silica substrates. Typical structures were defined through a two-layer lithographic process in which the deep region of the device (entropically favorable) was patterned and etched with one layer of lithography and the shallow region of the device (entropically unfavorable) was patterned and etched with a second layer of lithography. This is the configuration that others have used and modeled in the past. We have also made sideways-oriented devices using a one layer electron beam lithography process to define both the deep and shallow regions of the device. The devices are sealed by press bonding and annealing a 170um thick fused silica cover wafer to the substrate. Using both processes we have fabricated and tested devices with shallow regions as small as 20nm.

Other researchers have examined the trapping of DNA at entropic traps by averaging over many thousands of traps or by modeling the behavior of DNA in similar geometries [1-5]. Still others have examined DNA in two dimensional arrays of cavities separated by entropic barriers [4]. For investigating the dynamics of single molecules as they move through an entropic trap, the devices described herein offer a number of advantages over the aforementioned devices. The experimental setup and device geometry allow individual molecules to be observed throughout the trapping process with fast time resolution. Furthermore, the geometry used in these experiments allows for easy control of the length of the shallow region. This is important as the dynamics of the trapping-escaping process should depend upon the degree of confinement of the molecule throughout the process. The degree of control over the experimental parameters and the ability to observe individual molecules as they escape through entropic barriers yield information regarding the physical dynamics of the trap-escape process.

Some data are presented in Figure 3 for a sideways-oriented device with a 59nm wide shallow region. The data are fit to the theory presented by Han [5]. Devices with shallow regions of 20nm have been used to trap DNA molecules as short as 400 base pairs. With these dimensions, it should be possible to trap biomolecules with significant secondary or tertiary structure. Future work will focus on studying and separating large RNA complexes and proteins.

Figure 1: Experimental schematic. To measure the DNA trapping time, the DNA is illuminated and photons are collected.

Figure 2: A SEM showing a typical sideways entropic trap.

Figure 3: A graph of the trapping time versus the inverse electric field at the trap interface.

References:

[1] Entropic Trapping and Escape of Long DNA Molecules at Submicron Size Constrictions,

J. Han, S. W. Turner, H. G. Craighead, Physical Review Letters, 83, 1688 (1999).

[2] Mechanisms of DNA separation in entropic trap arrays: a Brownian dynamics simulation,

M. Streek, F. Schmid, T. T. Duong, A. Ros, Journal of Biotechnology, 112, 79 (2004).

[3] Electrophoretic Separation of Long Polyelectrolytes in Submolecular-Size Constrictions: A

Monte Carlo Study, F. Tessier, J. Labrie, G. W. Slater, Macromolecules, 35, 4791 (2002).

[4] Brownian Motion of DNA Confined Within a Two-Dimensional Array, D. Nykypanchuk,

H. H. Strey, D. A. Hoagland, Science, 297, 987 (2002).

[5] Characterization and Optimization of an Entropic Trap for DNA Separation, J. Han and

H. G. Craighead, Analytical Chemistry, 74, 394 (2002).