Concentrating Genomic Length DNA in a Microfabricated Array

We demonstrate that a microfabricated bump array can concentrate genomic-length DNA molecules efficiently at continuous, high flow velocities, up to $40\mu \mathrm{m}/\mathrm{s}$, if the single-molecule DNA globule has a sufficiently large shear modulus. Increase in the shear modulus is accomplished by compacting the DNA molecules to minimal coil size using polyethylene glycol (PEG) derived depletion forces. We map out the sweet spot, where concentration occurs, as a function of PEG concentration and flow speed using a combination of theoretical analysis and experiment. Purification of DNA from enzymatic reactions for next-generation DNA-sequencing libraries will be an important application of this development.

Figure 1

DNA concentrator using bump arrays with migration angle $\theta =3.8°$. (a) Schematic of the array. (The real array is 21 times longer than wide: approximately 3 cm long, 1.4 mm wide, $10\mu \mathrm{m}$ deep, and symmetric about the central wall.) DNA molecules enter via the inlet region, concentrate along the central (red) wall, and are collected at the product outlets. Red $\text{enlargement}=\text{array}$ of circular posts arranged in rows that are tilted towards the wall, which is also shown. All particles that follow the 3.8°-tilted rows of posts, have concentrated at this wall when they have flowed 1 cm into the array, for a net concentration of $\times 87$ before exiting the array. (b) Micrograph composite of purified 166 kbp T4 DNA in a solution with 10% PEG ($\mathrm{w}/\mathrm{v}$) in a flow with a peak speed of $30\mu \mathrm{mm}/\mathrm{s}$. The DNA concentrates along the central wall as it moves through the bump array.

Figure 2

Depletion force induced by PEG crowding. The centers of PEG molecules cannot come closer to a DNA strand than the radius of a PEG molecule. Thus each DNA molecule is surrounded by a zone that is depleted of centers of PEG molecules: PEG is restricted to the complement of these depletion zones. When depletion zones overlap, they take up less space, and hence their complement is larger. This increase in PEG-accessible volume increases the entropy of the PEG solution, which lowers its free energy. This causes an entropic force that favors increasing overlaps between depletion zones. At low number concentrations $c$ of PEG, the pressure that compresses overlapping depletion zones is $c{k}_{B}T$ [22]. This compression of depletion zones results in DNA compaction.

Figure 3

Purified 166 kbp T4 DNA in microfluidic array. (a) No PEG added and no flow. The DNA coils up to a spherelike object, slightly deformed by the posts. The concentric circles have radii of $R_g=1\mu \mathrm{m}$ and $2R_g$, respectively, with $R_g$ the estimated radius of gyration. (b),(c),(d) 0%, 5%, and 10% PEG concentrations, respectively, all at flow speed $20\mu \mathrm{m}/\mathrm{s}$. In (b) the DNA is elongated by the shear flow and reaches a length of $\sim 17\mu \mathrm{m}$, i.e., $\sim 30\%$ of its contour length. It follows the flow through the array. With PEG present, (c),(d), DNA is stretched less by the shear flow. At high PEG concentrations, DNA can maintain a globular conformation in the shear flow; hence, it behaves like a solid particle and is laterally displaced deterministically.

Figure 4

Heat map of the average extent along the flow for 166 kbp T4 DNA as a function of PEG concentration and peak flow speed or peak shear rate. This heat map is based on experimental data recorded at 30 points in the plane, those marked with circles in Fig. 5. Letters (a)–(d) refer to panels in Fig. 3.

Figure 5

Map showing which PEG concentrations and flow speeds or peak shear rates will concentrate 166 kbp T4 DNA (white area) or not (gray area) in the bump array in Fig. 3. The map is based on measurements done at the values marked with open circles. The transition between concentrated output or not is abrupt as a function of the PEG concentration and flow speed because of the large number of posts encountered by a molecule passing through the array [25]. Letters (b)–(d) refer to panels in Fig. 3.