Figure 1.
Schematic of the fabrication setup.
A computer-controlled custom current amplifier is used to apply voltages up to ±20 V and measure the current with sub-nA sensitivity from one of the two Ag/AgCl electrodes positioned on either sides of the membrane. It is noteworthy to realize that this experimental setup is identical (with the exception of the custom current amplifier replacing the commonly used Axopatch 200B) to the instrumentation used to study DNA or proteins translocation through nanopores.
Figure 2.
Nanopore formation by dielectric breakdown.
a) Application of a trans-membrane potential generates an electric field inside the SiNx, and charges the interfaces with opposite ions. b) Leakage current through the membrane follows a trap-assisted tunneling mechanism. Free charges (electrons or holes) can be produced by redox reactions at the surface or by field ionization of incorporated ions. The number of available charged traps (structural defects) sets the magnitude of the observed leakage current. c) Accumulation of charge traps produced by electric field-induced bond breakage or energetic charges carries leads to a highly localized conductive path, and a discrete dielectric breakdown event. d) A nanopore is formed following removal of the defects. e) Leakage current density for SiNx membranes (50-µm× 50-µm). The leakage current is fully reversible and stable, unless high fields are sustained, see Section S2 f) Leakage current at 5 V, on a 10-nm-thick SiNx membrane, in 1 M KCl at pH13.5. Pore created is ∼5-nm (18 nS). The slowly increase leakage current, following the capacitive spike, is a result of the accumulation of traps in the membrane. g) Experiment performed at 15 V, on a 30-nm-thick SiNx membrane, in 1 M KCl pH10. The nanopore is allowed to grow until a pre-determined threshold current is reached, at which point the voltage is turned off. The observed current fluctuations at the onset of pore formation are attributed to significant low-frequency noise at this voltage. Pore created is ∼3-nm (2.9 nS). h) Current-to-voltage curves for 3 nanopores fabricated on different membranes. The legend indicates the (pore diameter)/(membrane thickness) in nm. Measurements performed in 1 M KCl pH8, with an Axopatch 200B.
Figure 3.
Time-to-pore creation as a function of experimental conditions.
a) Semi-log plot of fabrication time of individual nanopores created in 30-nm-thick SiNx membranes in 1 M KCl buffered as indicated, versus the applied voltage and the calculated applied electric field. The number of separate nanopores each data point is averaged over is indicated in parentheses. The vast majority of nanopores plotted are sub-5-nm in size (i.e. <7 nS). b) Semi-log plot of fabrication time versus pH for the data plotted in a). c) Semi-log plot of fabrication time of individual nanopores created in 10-nm-thick SiNx membranes in 1 M Cl-based electrolyte buffered at pH 10 for different cationic species versus the applied voltage and the calculated applied electric field. All 66 nanopores plotted are sub-5-nm in size (i.e. <20 nS).
Figure 4.
a) Ionic current trace showing multiple DNA translocation events through a ∼6.4-nm pore in a 10-nm-thick SiNx membrane. Experiments performed with 10μg/mL of 5-kb DNA fragments in 3.6 M LiCl pH8, at 200 mV using an Axopatch 200B. Data sampled at 250 kHz and low-pass filtered at 100 kHz. b) Scatter plot of the normalized average current blockade (0% presenting a fully opened pore, and 100% a fully blocked pore) versus the total translocation time of a single-molecule event. Each data point represents a single DNA translocation event. The majority of the events are unfolded. There are very few anomalously long events, indicating weak DNA-pore interactions. The inset shows ionic current signatures of two single-molecule translocation events, passing in a linear and partially folded conformation. c) Histogram of the current level revealing the expected quantization of the amplitude of current blockades. Quantized levels corresponding to zero, one, two dsDNA strands in the nanopore are clearly observed.