Communicated by Robert H. Austin, Princeton University, Princeton, NJ, November 14, 2008 (received for review March 31, 2008)
Chip design. (A) Schematic of chip design showing microchannel arms connecting the circular reservoir pads to the nanoslit (yellow). (B) An enlarged 3D view of the nanopit lattice in the nanoslit with a cartoon DNA molecule (red) spanning 3 pits. (C) SEM images of 300 × 300 nm nanopits at high and low magnification.
Fluorescence micrographs of λ- and T4-DNA in arrays of 300 × 300 nm nanopits etched 100 nm deep with a surrounding 100-nm-deep slit. A–D show λ-DNA in arrays with lattice spacings of, respectively, 0.5 μm, 1.0 μm, 2.0 μm, and 5.0 μm. E–I show T4-DNA in arrays with lattice spacings of, respectively, 0.5 μm, 1.0 μm, 2.0 μm, 5.0 μm, and 10.0 μm. (Scale bar, 5 μm.) Note that in D, the true equilibrium conformation is in fact the single-pit state. The stretched configuration is metastable and decays on a timescale of minutes.
Image analysis protocol. (A) Example fluorescence image of λ-DNA in 300 × 300 nm nanopits with 1-μm spacing. The data analysis procedure involves integrating the fluorescence of boxes registered to the lattice (as shown). (B) Nanopit data channels for the molecule in A. The color of the data channels corresponds to the color of the boxes in A from which the data were obtained. The orange boxes are noise channels never occupied by the molecule during the course of the movie. A threshold can be applied to the integrated pit intensity to count the number of occupied pits as a function of time, shown in D. (C–E) The total number of filled pits as a function of time and representative fluorescence images taken 20 s apart for 300 × 300 nm nanopits with a 0.5-μm nanopit spacing (C), 1-μm nanopit spacing (D), and a 2-μm spacing (E). Insets show the histogrammed occupancy probability PN for the varying states accessed. (F) The histogrammed occupancy probability PN for the 300 × 300 nm nanopits with a spacing l = 0.5 μm. The red bars are the data for the molecule in C, the open bars with errors correspond to the mean values and standard deviations of PN for 14 molecules imaged in the 0.5-μm lattice and the blue bars are a theoretical prediction.
A 2D schematic of the nanopit arrays giving the definition of the pit spacing l, pit width a, the contour per pit Lp, and contour per linker Ls. The pit depth is denoted by d and the slit height by h.
Occupancy statistics. (A) The probability of a λ-DNA molecule occupying states with N = 1–8 as a function of lattice spacing l (h = 100 nm, a = 300 nm, and d = 100 nm). (B) The averaged occupancy for λ-DNA as a function of lattice spacing l. Error bars shown, which are comparable to the size of the data points, are the standard deviation of measurements over 10 molecules.
Metastable conformations. (A) The decay of a λ-DNA dimer to a monomer state for h = 100 nm, a = 300 nm, d = 100 nm, and l = 5 μm. Time runs from top to bottom with each image 0.24 s apart. The dimer state existed for about 50 s (only a portion of the movie around the decay event is shown). (B) The formation of a λ-DNA dimer state in a slit with h = 50 nm, a = 300 nm, d = 100 nm, and l = 10 μm. Time runs from top to bottom with each image 0.24 s apart. These states last in excess of 3 min. (C) The decay of a T4-DNA trimer state to a dimer with h = 50 nm, a = 300 nm, d = 100 nm, and l = 20 μm. Time runs from top to bottom with each image 0.45 s apart. The trimer state existed for 2 min before decaying. (D) A schematic illustration of the decay of a dimer state. Initially the molecule is suspended between 2 pits. The contour in each pit Lp fluctuates around the average 〈L2p〉 for the dimer configuration. A thermal fluctuation then empties the right pit and the linker recoils to the remaining filled nanopit which gains contour. Eventually, the molecule is left in a monomer state with the remaining nanopit containing contour 〈L1p〉. (E) The free energy as a function of nanopit contour Lp for a dimer and monomer state with a = 300 nm, d = 100 nm, and l = 5 μm (corresponding to the situation in A). The nanopit empties when a fluctuation drives Lp to the origin, corresponding to crossing the energy barrier Ub. Once Lp = 0, the molecule will recoil to the remaining nanopit and reach the monomer conformation (corresponding to the lower curve). (Scale bar, 5 μm.)
