Sorting by diffusion: an asymmetric obstacle course for continuous molecular separation

Abstract

A separation technique employing a microfabricated sieve has been demonstrated by observing the motion of DNA molecules of different size. The sieve consists of a two-dimensional lattice of obstacles whose asymmetric disposition rectifies the Brownian motion of molecules driven through the device, causing them to follow paths that depend on their diffusion coefficient. A nominal 6% resolution by length of DNA molecules in the size range 15-30 kbp may be achieved in a 4-inch (10-cm) silicon wafer. The advantage of this method is that samples can be loaded and sorted continuously, in contrast to the batch mode commonly used in gel electrophoresis.

Figure 1

Production of a sealed monolithic obstacle array.

Figure 2

A scanning electron micrograph of the obstacle course. The obstacles are 0.35 μm high and measure 1.5 × 6.0 μm. The gap between adjacent obstacles is 1.5 μm. An electric field propels the molecules directly through the gaps between the posts with velocity v. Transverse Brownian motion may cause a molecule to skip one channel to the right if it diffuses through displacement a_R, or very rarely, one channel to the left if it diffuses through a_L.

Figure 3

(A) The trajectories of XhoI-digested bacteriophage λ DNA fragments. The straight lines show the average electric field direction. The solid yellow line traces the path of a 33.5-kbp fragment; the purple line traces a 15-kbp fragment. The frame size is 130 × 114 μm². Total dimension of the separation device is 3 × 4 cm, and electric field strength is 1.4 V/cm. (A video clip illustrating the process is available at http://suiling.princeton.edu.) (B) Histogram of the deflection of approximately 200 DNA fragments of each size after they have passed through 14 gates (the vertical distance in A). Scale at bottom indicates the total number of channels that the molecule has shifted to the right. The solid lines are binomial distributions with the same mean and area as the experimental data.

Figure 4

Difference Δp in the probability per gate that 15-kbp and 33.5-kbp molecules skip to the right (blue). Extrapolated number of bands n_C that could be detected between 15 kbp and 33.5 kbp in a 10-cm sieve (red). Lines are theoretical predictions for Δp (blue) and n_C (red). The theoretical curve of Δp is taken from Eq. 1 of ref. , substituting in the two values of D/va, and calculating p_R and p_L for both fragments: Δp = [p_R(15 kbp) − p_L(15 kbp)] − [p_R(33.5 kbp) − p_L(33.5 kbp)]. Theoretical curve for band capacity n_C uses variance σ² = N [p_R(1 − p_R) + p_L(1 − p_L) + p_Rp_L)], given in ref. , where N = 12,500 gates.