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) [1] 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. [2,3] The a-HL nanopore has been used to study the unzipping kinetics of DNA hairpin molecules under stationary or time-varying forces, [4] to detect DNA hybridization kinetics, [5] and to study the interaction of DNA with bound proteins using nanopore force spectroscopy. [6] In addition, a-HL can be biochemically modified for various sensing tasks, such as analyte detection and ligand-receptor interactions.[7]Solid-state nanopores can be fabricated in thin Si 3 N 4 and SiO 2 membranes, using either an Ar + beam [8,9] or an electronbeam (e-beam) in a transmission electron microscope (TEM), [10] as well as in a variety of materials using other techniques.[11] 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. [12,13] 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, [14] 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 Si 3 N 4 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). [15,16] Investigation of pore contraction/expansion dynamics [17] 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...

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106 V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.

The effect of bacterial motion on the diffusion of a molecule of high molecular weight is studied by observing the mixing of two streams of fluid in a microfluidic flow cell. We show that the presence of motile E. coli bacteria in one of the streams results in a marked increase in the effective diffusion coefficient of Dextran, which rises linearly with the concentration of bacteria from a baseline value of 0.2×10−7 to 0.8×10−7 (cm2/s) at a concentration of 2.1×109/ml (approximately 0.5% by volume). Furthermore, we observe that the diffusion process is also observed to undergo a change from standard Fickian diffusion to a superdiffusive behavior in which the diffusion exponent rises from 0.5 to 0.55 as the concentration of bacteria rises from 0 to 2.1×109/ml.

Solid-state nanopores can be used to detect nucleic acid structures at the single molecule level. An e-beam has been used to fabricate nanopores in silicon nitride and silicon dioxide membranes, but the pore formation kinetics, and hence its final structure, remain poorly understood. With the aid of high-resolution TEM imaging as well as TEM tomography we examine the effect of Si 3 N 4 material properties on the nanopore structure. In particular, we study the dependence of membrane thickness on the nanopore contraction rate for different initial pore sizes. We explain nanopore formation kinetics as a balance of two opposite processes: (a) material sputtering and (b) surface-tension-induced shrinking.