Rapid Fabrication of Uniformly Sized Nanopores and Nanopore Arrays for Parallel DNA Analysis

Abstract

Nanometer-sized pores can be used to detect and characterize biopolymers, such as DNA, RNA, and polypeptides, with single-molecule resolution. Experiments performed with the 1.5 nm pore a-hemolysin (a-HL) demonstrated that singlestranded DNA and RNA molecules can be electrophoretically threaded through a pore, and that the ion current flowing through the pore contains information about the biopolymer sequence: its type, length, and secondary structure. The a-HL nanopore has been used to study the unzipping kinetics of DNA hairpin molecules under stationary or time-varying forces, to detect DNA hybridization kinetics, and to study the interaction of DNA with bound proteins using nanopore force spectroscopy. In addition, a-HL can be biochemically modified for various sensing tasks, such as analyte detection and ligand–receptor interactions. Solid-state nanopores can be fabricated in thin Si3N4 and SiO2 membranes, using either an Ar beam or an electron beam (e-beam) in a transmission electron microscope (TEM), as well as in a variety of materials using other techniques. Solid-state nanopores offer several advantages over phospholipid-embedded protein channels, namely, their size can be tuned with nanometer precision and they exhibit an increased mechanical, chemical, and electrical stability. Recent studies using solid-state pores have begun to emerge, demonstrating the detection of single-stranded and double-stranded DNA molecules. A major advantage of solid-state nanopores is that they can, in principle, be integrated into devices compatible with other detection schemes in addition to ion current measurements. In particular, optical-based methods offer straightforward parallelism through the simultaneous probing of many nanopores. Optical methods for sensing single molecules can be implemented by labeling the biomolecules and/or the nanopores. Although protein pores embedded in a phospholipid bilayer can be interrogated optically to detect single molecules, a stable, long-timescale probing is very complicated since the pores readily diffuse in the bilayer, leading to aggregation and destabilization of the membrane. In contrast, nanopores fabricated in solid-state materials are static, and are therefore more compatible with optical probing. In this paper, we extend state-of-the-art techniques by demonstrating the rapid fabrication of finely tuned nanopores and nanopore arrays. The nanopores were fabricated in thin Si3N4 films using the intense e-beam of a field-emission TEM. By maximizing the e-beam density at the specimen we achieved a nearly fivefold decrease in the fabrication time of a single nanopore (ca. 30 s). Investigation of pore contraction/expansion dynamics under different irradiation conditions enabled nanopore fabrication in the range of 2–20 nm with exceptional size control (<0.5 nm variability). Since the nanopores were fabricated sequentially (i.e., using one e-beam), both the reduction in fabrication time and size control were crucial for the manufacturing of nanopore arrays. The 3D nanopore shape was extensively characterized by performing TEM tomography, as well as by ion-current measurements through the pores. Finally, the detection of double-stranded DNA molecules through 4 nm diameter nanopores was demonstrated by monitoring their translocation under an applied bias. The starting materials for TEM processing were either fabricated in house or by Protochips Inc. (Raleigh, NC), using the following procedure: low-pressure chemical vapor deposition (LPCVD) was used to form a Si3N4 film (20 or 50 nm thick) on one side of a 500 lm thick Si wafer. A 100 lm× 100 lm window was then fabricated in the wafer using photolithography and standard wet-etching. Nanopore fabrication was then carried out in the thin Si3N4 membrane using a JEOL 2010F field-emission TEM. Alignment of the e-beam involved the adjustment of the condenser stigmatism to the familiar triangle-shaped beam, using a large condenser aperture. The resulting electron-energy-density distribution displayed a threefold aberration. The condenser lens was then used to fully converge the beam to an intense point with a triangular halo of low intensity. The column was then aligned using standard high-resolution transmission electron microscopy alignment procedures. After the alignment procedure, nanopores with diameters (d) in a range from 3 to 6 nm were directly drilled using an e-beam intensity of ca. 2.5× 10 e nm and a CO M M U N CA IO N