Joshua Cross, Elizabeth Strychalski
Laser induced fluorescence and photon burst counting were used to measure the time individual lambda DNA molecules were delayed at entropic barriers in nanofluidic channels. In this work, we bring experimental techniques designed for quantitative single molecule studies to bear on the observation of individual DNA molecules as they are delayed by, probe, and finally overcome entropic barriers. The sizes of DNA molecules and their flow speeds in fluidic channels can be quantified by measuring photon bursts collected when fluorescently labeled molecules pass through a focused laser [1,2]. Our ability to carefully measure the delay time for individual molecules represents an improvement over the indirect, or bulk, methods previously used to determine the same quantity .
Our devices are fabricated on a fused silica substrate to which a 170um thick piece of fused silica is bonded. The fabrication involves two layers of aligned lithography and subsequent reactive ion etching to form the non-restrictive and restrictive regions of the device. The restrictions represent entropically unfavorable regions for DNA molecules. The rate of passage through these restrictive regions has previously been shown to depend upon the size of the molecule [3-6], and these so-called entropic traps have been used to fractionate DNA samples based upon molecular length .
Previous measurements of the entropic trapping time with geometries similar to ours were determined by averaging over the trapping times caused by many traps along an array . Others have measured diffusion times and jumping frequencies for DNA confined within a two dimensional array of cavities separated by entropic barriers, but their work was done for traps shorter than the contour length of the molecule and with time resolution longer than the time it takes the molecules to make the jump . In this work we use photon burst counting to determine the trapping time for individual molecules as they encounter individual entropic traps. By measuring the dependence of the trapping time on the molecular size, the cross sectional dimensions of the entropic trap, and the length of the entropic trap, we hope to refine our understanding of DNA motion through entropically unfavorable regions. Specifically, by characterizing the trapping time as a function of device and molecule parameters, we should be able to optimize the entropic trap device to achieve higher resolution and faster molecular separations than achieved by Han et al . Furthermore, the fused silica fabrication method described herein enables trapping regions with much smaller dimensionswe have tested devices with 20nm restrictionsand this may lead to efficient separation of even smaller biomolecules than reported previously.
Figure 1. Schematic side view of an entropic trap and the unrestrictive channel on either side of the trap.
Figure 2. This figure shows the number of photons detected versus time. The width of a particular burst indicates the duration a molecule is resident in the focal volume.
Figure 3. Measurements of the trapping time of lambda DNA as it probes an entropic trap. At lower field strengths the trapping becomes significant.
1. High-throughput flow cytometric DNA fragment sizing, A. Van Order, R.A. Keller, W.P. Ambrose, Analytical Chemistry, 72, 37 (2000).
2. DNA fragment sizing by single molecule detection in submicrometer-sized closed fluidic channels, M. Foquet, J. Korlach, W. Zipfel, W.W. Webb, H.G. Craighead, Analytical Chemistry, 74, 1415 (2002).
3. 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).
4. "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).
5. Electrophoretic Separation of Long Polyelectrolytes in Submolecular-Size Constrictions: A Monte Carlo Study, F. Tessier, J. Labrie, G.W. Slater, Macromolecules, 35, 4791 (2002).
6. Characterization and Optimization of an Entropic Trap for DNA Separation, J. Han and H.G. Craighead, Analytical Chemistry, 74, 394 (2002).
7. Brownian Motion of DNA Confined Within a Two-Dimensional Array, D. Nykypanchuk, H.H. Strey, D.A. Hoagland, Science, 297, 987 (2002).