Nanopore sensor has the advantage of both high-throughput and low-cost compared to other traditional methods for characterization of single molecules. In a typical nanopore meas-urement, biomolecules are electrophoretically driven through a nanopore one at a time by an external electric field, due to the physical occupation as well as the strong interaction between the molecules and the nanopore wall, the modulated ionic current can reflect the information, including charge state, shape, orientation and so on, carried by the molecules. Thus, nanopore sensor has been widely used to detect and characterize DNA molecules, single proteins, pro-tein-DNA complexes and even single nucleotide in a single DNA strand and single amino acid in a single peptide. However, there remain some challenges in the area of detecting DNA and protein molecules due to no consensus on the ionic current signal that is related to the corre-sponding molecule, as the charge state, the orientation and secondary structure of the molecule will all induce various blockade currents. To enrich the fundamental research in the area of sin-gle molecule detection by nanopores and facilitate the development of nanopore sensor, in this thesis, we mainly focused on the nucleic acid molecules and protein molecules detection by us-ing nanopores and studied the mechanism of current modulation theory. We firstly investigated the DNA transport dynamics through both solid-state nanopores and biological nanopores. Then the mechanism of nanopore access resistance was studied by using atomic force microscopy and we also realized slowing down DNA transport through solid-state nanopores and manipulation of DNA translocation direction by using atomic force microscopy. Finally, we used nanopore to characterize protein folding states, protein folding-unfolding transitions and protein folding in-termediates. Our main results and conclusions obtained by using both theoretical and experi-mental methods are summarized as below:
1. Using molecular dynamics simulations, graphene nanopores were used to investigate dsDNA transport dynamics through nanopores and to differentiate the ssDNA homopolymers composed of different nucleotides. The translocation speed of a dsDNA through the graphene nanopore could be controlled by adjusting the appropriate applied bias and the diameter of the nanopore. A smaller bias could slow down the dsDNA translocation through the nanopore due to the small electric force acting on the dsDNA. It was also found that the dsDNA translocation speed decreased with the nanopore diameter, it is because the potential drop across a larger na-nopore is a bit smaller compared to a smaller nanopore. Molecular dynamics simulations were also performed to study the translocation process of four ssDNAs with ten identical bases through graphene nanopore with diameter of 2 nm. Due to the similar dimension of the four nu-cleotides, the blockage current was unlikely to provide a distinguishable signal for the homo-polymers. However, by simply monitoring and analyzing the translocation time of poly(dA)10, poly(dC)10, poly(dG)10 and poly(dT)10 though the nanopore, each ssDNA could be identified and characterized.
2. As found above that the nanopore dimension could influence the translocation speed of DNA through the nanopore, to validate this interesting finding, we investigated the nanopore size effect on translocation of poly(dT)30 through Si3N4 membrane using experimental method. It was found that the speed of the poly(dT)30 transport through Si3N4 nanopores can be slowed down by half through increasing the nanopore diameter from 4.8 nm to 10.8 nm. The results were consistent with our simulation results. Besides, the current blockage induced by DNA passing through the nanopore was less obvious as pore diameter is larger, which is in good agreement with the theoretical prediction.
3. The electrophoretic transport mechanism of flexible DNA homopolymer composed of thymines through the α-hemolysin nanopores in high concentration potassium chloride solution was studied. Two obvious current blockades were found to be induced by poly(dT)20 transloca-tion and collision events. Both blockade currents increase linearly with the applied bias. How-ever, the relative blockade currents are almost kept the same though variable voltages were ap-plied. The collision times of poly(dT)20 in the luminal site of the pore remain constant for dif-ferent voltages. The translocation speed of poly(dT)20 through the nanopore decreases as the applied bias increases. It is because as the potential increases, the drag force acting on the ho-mopolymer is much easier to help it crumple into a cluster due to the poor stacking of thymine residues compared to homopolymers consisting of other nucleotides. Molecular dynamics simu-lations further confirmed the experimental results.
4. The current modulation mechanism is significant for designing the single molecule sen-sor based on nanopores. In this thesis, we studied how an occluding object placed near a na-nopore affects its access resistance by integrating a nanopore sensor with an atomic force mi-croscopy. It was found that there exists a critical hemisphere around the nanopore, inside which the tip of an atomic force microscopy would affect the ionic current. The radius of this hemi-sphere, which is a bit smaller than the theoretical capture radius of ions, increases linearly with the applied bias voltage and quadratically with the nanopore diameter, but is independent of the operation modes and scanning speeds of the atomic force microscopy. A theoretical model was also proposed to describe how the tip position and geometrical parameters affect the access re-sistance of a nanopore.
5. A measuring system that integrates nanopore sensor with atomic force microscopy was designed to control DNA translocation through solid-state nanopores. By attaching the dsDNA strand to the AFM probe tip using thiol-Au or biotin-streptavidin interaction, we can slow down DNA translocation speed to 2 ?/ms, this is slow enough to gain sufficient data for extracting structure information as the bandwidth for the ionic current measurement has reached to 5 MHz by now. Another advantage of the designed measuring system is that, we can realize even reversing the DNA translocation direction and repeatedly dragging out the DNA strand and threading it through the nanopore for many times as long as the DNA is not exfoliated from the AFM probe tip.
6. Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states - attractive pharmaceutical targets. Although conventional single-molecule approaches can detect folding intermediates, they presently lack throughput and require elaborate labeling. Here, we theoretically show that measurements of ionic current through a nanopore containing a protein can report on the protein’s folding state. Our all-atom molecular dynamics simulations show that the unfolding of a protein lowers the nanopore ionic current, an effect that originates from the reduction of ion mobility in proxim-ity to a protein. Using a theoretical model, we show that the average change in ionic current produced by a folding?unfolding transition is detectable despite the orientational and con-formational heterogeneity of the folded and unfolded states. By analyzing millisecond-long all-atom MD simulations of multiple protein transitions, we show that a nanopore ionic current recording can detect folding?unfolding transitions in real time and report on the structure of folding intermediates.