Single-file electrophoretic transport and counting of individual DNA molecules in surfactant nanotubes

Abstract

We demonstrate a complete nanotube electrophoresis system (nanotube radii in the range of 50 to 150 nm) based on lipid membranes, comprising DNA injection, single-molecule transport, and single-molecule detection. Using gel-capped electrodes, electrophoretic single-file transport of fluorescently labeled dsDNA molecules is observed inside nanotubes. The strong confinement to a channel of molecular dimensions ensures a detection efficiency close to unity and identification of DNA size from its linear relation to the integrated peak intensity. In addition to constituting a nanotechnological device for identification and quantification of single macromolecules or biopolymers, this system provides a method to study their conformational dynamics, reaction kinetics, and transport in cell-like environments.

Fig. 1.

Schematics describing nanotube-vesicle network (NVN) formation, geometry of the NVN system, and fluorescence detection used in the experiments and simulations of the field distribution and flow profile. (a) The electrodes are pressed against the membrane of the unilamellar liposome filled with a DNA solution, and an electric pulse is applied. The pulse opens pores in the membrane, allowing the tips of the electrodes to enter into the liposome. (b) The pores in the membrane close, and the membrane seals around the electrode. (c) One of the electrodes is moved out of the unilamellar liposome, pulling a lipid membrane nanotube. (d) Schematic picture of the NVN. The nanotube is aligned across the confocal excitation/detection spot of the confocal microscopy setup, and an electric potential is applied between the electrodes, with the nanotube-coupled electrode having positive potential. The fluorescence signal from the DNA molecules is detected as they pass the excitation/detection spot. V₁ is the velocity of the DNA molecules, V₂ is the velocity of the membrane, and V₃ is the velocity of the electroosmotic flow. Because V₂ and V₃ cancel each other out (see text), no net liquid flow occurs in the nanotube. (Inset) The equivalence circuit of the electrophoretic system is shown; 1 and 5 represent the electrode resistance (50 MΩ), 2 and 3 represent the vesicle-electrode seal resistance (50 MΩ) and the nanotube-electrode seal resistance (100 MΩ), respectively, and 4 represents the nanotube resistance (>1GΩ), calculated from the buffer conductivity (0.5 S/m) and the nanotube dimensions (L = 40 μm and R = 150 nm). Evaluation of the equivalence circuit gives a potential drop of 60 mV over the nanotube. (e) A Nomarski differential interference contrast microscopy image of a fully formed NVN with electrodes. The unilamellar vesicle is filled with fluorescently labeled DNA molecules. The two electrodes are used both to create the NVN and drive the electric current through it. The length of the nanotube is 40 ± 1 μm. (f) Field strength distribution inside and outside the NVN, modeled with the finite element method program femlab 3.0.(g) Flow profile of axial velocity inside the nanotube, drawn from the nanotube center line to the membrane surface.

Fig. 2.

Electropherograms of DNA from φX174 (a), pΔ (b), T7/BsteII (c), T7 (noncleaved) (d), and T4 (e) (compare Table 1). The DNA molecules were transported through a 40-μm-long lipid membrane nanotube and detected at its midpoint. The effective field strength was ≈15 V/cm in all experiments.

Fig. 3.

Distributions of peak intensity, transit time, and integrated peak area for the different DNA species. Distributions for T4 DNA are not shown because of the small number of observed events (n = 12). (a-c) Distributions of peak intensity (a), transit time (b), and integrated peak area (c) for DNA from φX174, pΔ, T7/BsteII, and T7 (noncleaved) are shown. (d-f) Distributions of peak intensity (d), transit time (e), and integrated peak area (f) for the linear and relaxed circular forms of φX174 DNA. The distributions for T4 DNA are not shown because of the rarity of the detected peaks compared with the other DNA sizes (for which n > 100); only average values (n = 12) of the three parameters are given in Fig. 4.

Fig. 4.

Mean values of integrated peak area (A_I) (a), peak intensity (I_P) (b), and transit time (Δt) (c) vs. DNA size (N). Error bars correspond to the standard error. Where the error bars are not clearly visible, they are equal to or smaller than the data points.