Defense Date


Document Type


Degree Name

Doctor of Philosophy


Physics and Applied Physics

First Advisor

Dr. Joseph E. Reiner

Second Advisor

Dr. Daeha Joung

Third Advisor

Dr. Michael A. Reschikov

Fourth Advisor

Dr. Soma Dhakal


There have been significant advances in detecting and characterizing small molecules using a new real-time single-molecule sensing nanopore technology. But still, there remains a significant challenge with the biomolecules comprised of several amino acids such as enzymes, peptides, metabolites, proteins, etc., due to their complex structures and charge distribution. Unlike nucleic acids, every amino acid is unique in size, polarity, charged side chains, hydrophobicity, structure, and conformation resulting in non-uniform or complex interaction within the nanopore, making sensing complicated. In addition, small molecule sensing through a nanopore is made difficult because of the shorter-lived and smaller-magnitude current blockades that result. The first segment of the thesis details a novel approach to detecting molecules outside the pore at a single cluster limit to overcome these issues. Water-soluble ligand capped gold clusters are trapped in a pore lumen for several seconds under applied transmembrane potential, which rapidly senses thiol-containing ligands interacting on the cluster surface via a ligand exchange process. The exchange process leads to unique time-resolved current transitions proportional to their mass (volume). The dissertation further introduces the characterization and detection of several cysteine selective small peptides (<1 kDa) that undergo an exchange process with the tiopronin capped cluster trapped in a nanopore. The exchange phenomena give rise to either well-resolved transitions between unique current states or high-frequency two-state fluctuations dependent on the peptide’s size, the position of a cysteine residue in the sequence, or the overall net charge of the peptide.

Understanding that the molecular dynamics of the analyte can be studied using cluster-modified alpha hemolysin (αHL) nanopores, we have further explored our research on dynamic Holliday junction (HJ) DNA molecules to monitor their kinetics and conformational states near the vicinity of the pore. A single HJ molecule that constantly switches between isomer states (iso-I, iso-II) and intermediate open state is captured in a pore lumen under an applied electric field, leading to rapid, two-state, fluctuations in the current. We specifically focused on understanding the HJ’s conformational state kinetics, which is essential for biological and non-biological processes such as homologous recombination, DNA repair, and as a target for therapeutics, nucleic acid sensing, and designing smart DNA devices. Moreover, we have demonstrated that toehold mediated HJs can be developed as a highly selective sensor capable of detecting short oligo DNA strands.

The results obtained in this thesis describe a new way of detecting water soluble peptides through cluster-modified αHL nanopores. The goal of this approach is to develop a biosensor for early detection of cysteine-containing cancer marking peptides. The tools developed in this portion of the thesis have been further applied to study previously inaccessible high frequency (> 100 Hz) dynamics of nano-confined HJ molecules. This opens the door to studying DNA-protein interactions at timescales that more closely resemble in vivo conditions and further enables the development of a sensor for a wide variety of oligonucleotide targets.


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Available for download on Sunday, May 02, 2027