DNA and ion transport through solid-state nanopores

This thesis describes experimental work on a novel type of devices capable of detecting single-(bio)molecules; nanometer-sized pores, or nanopores. Individual nanopores are placed in between two electrolyte-filled liquid compartments and (bio)molecules are electrophoretically driven through them. During passage of a single (bio)molecule the ionic current is temporarily blocked, as part of the liquid volume carrying the current is now occupied. In this thesis, we report on the fabrication and characterization of solid-state nanopores, and their use in single-molecule translocation studies. We first describe the fabrication of solid-state nanopores using a high-intensity electron beam in a transmission electron microscope. Focussing the beam on thin free-standing membranes creates nanopores of controlled sizes, down to 0.4 nm radius. We examine their geometry and local surface composition and identify a material-dependent response to the electron beam. Nanopores fabricated in pure SiN membranes show much smaller depletion areas compared to nanopores fabricated in SiO2/SiN/SiO2 membranes. Furthermore, we demonstrate that the sidewall angle and the local material composition near a nanopore in SiO2 containing membranes can be influenced using electron beams of different intensities. Subsequently, we study ionic current fluctuations in our fabricated solid-state nanopores. At low frequencies (ƒ ≤ 100 Hz) we observe 1⁄–type of noise, with strong pore–to–pore variations. First, we consider nanopores that do not show excessive low–frequency noise. We show that their noise power remarkably scales linearly with the inverse number of charge carriers, in agreement with Hooge's relation. We find a Hooge parameter α = (1.1 ± 0.1) ·10-4. Next, we examine the excessive low–frequency noise by scanning nanopores through the focus of an infrared laser beam and recording the ionic current through them simultaneously. We show a correlation between a decreased nanopore conductance and increased amount of ionic current fluctuations. In addition, we measure an unexpected double–peak ionic conductance profile which can be explained by a simple model of a cylindrical nanopore containing a nanometer–sized gaseous bubble (nanobubble). We infer that nanobubbles may act as a source of excessive low–frequency noise in solid–state nanopores. Finally, we use solid–state nanopores to measure ion, double strand (ds)DNA and fully protein–coated dsDNA transport. The ionic conductance shows a three–order–of–magnitude decrease with decreasing salt concentrations from 1 M to 1 μM, strongly deviating from bulk linear behavior. We successfully model the data by accounting for a salt–dependent surface charge of the nanopore. Double stand DNA translocation in salt concentrations ranging from 50mM to 1 M is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0:4 M). The data are described by a model where current decreases result from the partial blocking of the pore due to the dsDNA area, and current increases are attributed to the motion of the counter ions screening the DNA backbone charge. We infer an effective dsDNA charge of 0.58 ± 0.02 electron charges per basepair. Translocation measurements on fully recombination protein A (recA)–coated dsDNA molecules are shown to result in current blockade events with a wide variety in time duration (≈ 10-4 – 10-1 s) and conductance blockade values (2.5 – 14 nS). We identify the events with high conductance blockades (11.9 ± 0.5 nS) with the translocation of dsDNA coated with recA and we estimate a 8.5 nm diameter for the nucleoprotein filament. Furthermore, we present the rate of RecA–coated dsDNA current blockade events as a function of voltage, and find an exponential dependence at low voltages and a constant rate at high voltages. In conclusion, we have used tools from nanotechnology to fabricate solid–state nanopores. These nanometer–sized pores are characterized by studying ionic current fluctuations. We use the nanopores experimentally to detect and measure the transport of ions, DNA and fully protein–coated DNA molecules. The obtained results contribute to the development, understanding and use of the recently introduced solid–state nanopores as single–molecule (bio)sensors.Applied Science