Nanopore Based Single-molecule Sensors
Abstract
In the past two decades nanopores have been used as highly sensitive
detection systems for exploring the properties of analytes at single molecule
resolution. The small dimensions of a nanopore permit the molecule of interest to
be confined within it, allowing for the extraction of valuable information relating to
its physical and chemical properties. Single molecule analysis, as opposed to bulk
measurements does not involve ensemble averaging. Hence, short-lived states
such as an intermediate configuration during a conformational change can be
observed directly, while such states would be masked in the bulk assay.
The main project described in this thesis involves the design and fabrication
of a hybrid silicon nitride-DNA origami nanopore system for use in biosensing of
proteins. We used the nanopore system to experimentally observe the effect of
forces between the translocating molecule and nanopore with a focus on the
electro kinetics inside the pore and escape rate problem. These are further verified
by finite element simulations and MATLAB simulations which enables us to
investigate the physics behind the different types of events that we observe.
The key findings from this work can be summarized as follows. We report
on an operating regime of this nanopore sensor, characterized by attractive
interactions between the nanoparticle and the pore, where the dwell time is
exponentially sensitive to the target-pore interaction. We used negatively and
positively charged gold nanoparticles to control the strength of their interaction with
the negatively charged silicon nitride pore. Our experiments revealed how this
modulation of the electrostatic force greatly affects the ionic current with an
exponential dependance of dwell times. A stochastic model is developed for
analyzing this analyte-pore interaction based on the well-known Kramer’s problem
of escape from a barrier.Finally, the nitride nanopore was functionalized using DNA
origami with thrombin binding aptamer (TBA15), a well studied 15-mer aptamer
DNA sequence that binds selectively with thrombin protein. Consistent with our
previous experiment, we observed current traces with large dwell time blockades
for thrombin whereas for another protein the trace contained minimal dwell time current enhancements. The presence of TBA15 aptamer increased the interaction
energy between the thrombin and the nanopore resulting in a blockage with
comparatively larger dwell time and enabled us in sensing thrombin at
concentrations as low as 20nM.
Nanopore technology will remain an important field of science in the
21st century. We believe equipped with our understanding of nanopore analysis, in
future we will be able to detect and unravel important physical phenomena in the
single molecule world.