| Current single molecule DNA sequencing technologies require extensive biochemical sample amplification. These methods introduce biases and lose epigenetic information, preventing full exploitation of DNA sequencing technology. Single Molecule, Real-Time (SMRT) sequencing, the technique used in the work described in this thesis, requires DNA amplification due to inefficiency in the DNA loading process prior to sequencing. This inefficiency arises from an entropic barrier to DNA entrance into the confined sequencing volume, which is a ~100 nm cylindrical aperture, referred to as a zero-mode waveguide (ZMW). We propose overcoming this barrier by placing a nanopore at the waveguide base, creating a nanopore-zero-mode waveguide (NZMW). The nanopore is a ~1-10 nm hole in a ~30 nm-thick insulating film, which may be used to electrophoretically attract charged molecules like DNA. Nanopores may be made in a variety of dielectric materials, usually silicon nitride (SiN). When biased, this pore establishes a DC electric field that attracts DNAs. A molecule may traverse the pore itself, in a process called translocation, inducing a transient, discrete drop in nanopore conductance. Measuring the rate of these current spikes allows one to measure the rate of capture of polymers by the nanopore. This system allows efficient molecular capture at low concentrations. This implies that, with efficient molecular capture an NZMW could use significantly lower concentrations of sequencing sample molecules. This paves the way for low-amplification or amplification-free sequencing.;On the way to fabricating, characterizing, and utilizing the NZMW for sequencing, we investigate the capture of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and proteins by nanopores. We develop hafnium oxide as an alternative material for nanopore membranes and demonstrate its high stability and strong interaction with translocating DNA molecules. Further, using high bandwidth electronics, we detect fast, ~micros-long protein translocation events, and establish solid-state nanopores as an excellent tool for measurements of single, unlabeled proteins. Furthermore, we show that, when confined in the nanopore, proteins interact with the nanopore surface, drastically reducing their diffusion compared to free solution. Finally we outline the fabrication and characterization of an NZMW device and show its ability to capture biomolecules. We close by demonstrating efficient capture and sequencing of template DNAs, including long, 20, 000 base pair fragments. |
