Transverse dielectrophoretic-based DNA nanoscale confinement

Abstract

Confinement of single molecules within nanoscale environments is crucial in a range of fields, including biomedicine, genomics, and biophysics. Here, we present a method that can concentrate, confine, and linearly stretch DNA molecules within a single optical field of view using dielectrophoretic (DEP) force. The method can convert an open surface into one confining DNA molecules without a requirement for bonding, hydrodynamic or mechanical components. We use a transverse DEP field between a top coverslip and a bottom substrate, both of which are coated with a transparent conductive material. Both layers are attached using double-sided tape, defining the chamber. The nanofeatures lie at the "floor" and do not require any bonding. With the application of an alternating (AC) electric field (2 V_p-p) between the top and bottom electrodes, a DEP field gradient is established and used to concentrate, confine and linearly extend DNA in nanogrooves as small as 100-nm in width. We also demonstrate reversible loading/unloading of DNA molecules into nanogrooves and nanopits by switching frequency (between 10 kHz to 100 kHz). The technology presented in this paper provides a new method for single-molecule trapping and analysis.

Figure 1

Schematic of the device shows (a) two parallel plate electrodes separated by a spacer defining the chamber. DNA (blue) is located inside the chamber in a free solution before applying electric field; (b) nanoscale confinement of DNA molecules with an applied AC field creating a transverse dielectrophoretic force; (c) numerical model of the chamber (with vertical chamber spacer height of 30 μm) showing the transverse electric field lines (red) from the top to bottom electrode; (d,e) fluorescence image showing DNA molecules inside the chamber before and after applying the electric field. (e) With a transverse DEP force, DNA is confined and linearly stretched inside 300-nm channels. (f) Image of a fused silica prototype.

Figure 2

Schematic diagram showing the model geometry with electrodes, inlet/outlet, insulator and channel wall; (b) surface map showing the normalized electrical field strength for the applied voltage of 2 V; (c) simulation showing the accumulation of molecules due to the negative DEP field. The surface map of electric field gradient (grey) is superimposed with particle (yellow) accumulation and (d) fluorescent image showing DNA concentration of λ-Phage DNA with application of DEP force.

Figure 3

Clausius-Mossotti function k (equation 2) plot showing cross-over between positive to negative DEP and the crossover frequency.

Figure 4

(a–d) DNA build-up along an electrode upon ramping frequency from 10–200 kHz (2 Vpp). (a) DNA in free solution starts to move; (b) upon application of DEP, molecules start to move towards the electrode (in image center); (c,d) molecules eventually become highly concentrated along the electrode.

Figure 5

Normalized electrode intensity versus frequency showing molecule focusing along the electrode.

Figure 6

(a) Schematic of DEP-assisted DNA (green) confinement and linearization on nanoscale grooves; (b) SEM image of nanogroove electrodes. Fluorescent image of λ-phage DNA on (c) 1-μm and (d) 300-nm nanopatterned electrodes showing parallel confinement and extension.

Figure 7

(a) Time series images of λ-phage DNA extension in nanoscale confinement with DEP force. (b) and (c) λ-phage DNA confinement and extension on (b) 300-nm and (c) 100-nm electrodes. (d) Probability density of λ-phage DNA length at several time frames, and (e) λ-phage DNA extension in different electrode dimensions.

Figure 8

Intensity plot across linearized DNA for 300-nm and 100-nm channel and background signal intensity.

Figure 9

Comparison of the DNA extension length with the geometric average dimensions plotted in log-log scale. Circles are data of conventional confinement extension from the literature^,, and diamonds are DEP-based extension.

Figure 10

(a) Schematic diagram of DEP-assisted DNA confinement on nanoscale pits, (b) SEM image of the device with nanopatterned pits. (c) Fluorescence image showing DEP-assisted λ-phage DNA confined in nanopits (bright spots). (d) The intensity profile shows sharp peaks at confined DNA molecules inside nanopits.

Figure 11

Fabrication flow (a) transparent conductive layer (Indium Tin Oxide) coating on fused silica substrate, (b) deposition of insulator layer, (c) reactive ion etching of insulator layer using E-beam lithography mask, (d) formation of flow chamber by joining top and bottom layer with double-sided tape, (e,f) AFM surface morphologies of electrode surface and insulator (SiNx) surface showing smooth ITO surface with roughness RMS of 1.81 nm, and (g) SEM image of fabricated nanochannel on the insulator layer.