Graduation Semester and Year
Fall 2025
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Biomedical Engineering
Department
Bioengineering
First Advisor
Dr. George Alexandrakis
Second Advisor
Dr. Minjun Kim
Third Advisor
Dr. John Weidanz
Fourth Advisor
Dr. Weidong Zhou
Abstract
The rapid advancement of nanomedicine has enabled increasingly precise nanotherapeutic platforms, including targeted nanoparticles, monoclonal antibodies, and viral gene delivery vectors. As these technologies mature, quality control and analytical characterization have emerged as critical bottlenecks, particularly in systems where functional outcomes depend on one-to-one molecular or particle–biological interactions. Conventional ensemble-averaged techniques obscure heterogeneity that is often central to therapeutic efficacy, safety, and manufacturability. A prominent example is adeno-associated virus (AAV)–based gene therapy, where production yields heterogeneous populations of empty, partially loaded, and fully loaded capsids. Genome fill percentage and integrity directly influence therapeutic efficacy and dosing accuracy, while limited understanding of DNA organization within intact AAV particles further constrains analytical interpretation. ii Recent label-free single-particle techniques, including mass photometry and solid-state nanopores, have begun to address these challenges but remain limited in resolving partially loaded AAV populations. Mass photometry enables direct mass measurement of individual capsids yet struggles when genome differences in internal DNA organization produce minimal mass contrast. Similarly, conventional nanopore measurements often yield overlapping current blockade signatures for empty, partially loaded, and full capsids due to their similar size, charge, and geometry, limiting reliable classification of under-filled populations. This dissertation addresses these limitations by developing plasmonic nanopore methodologies that combine simultaneous optical and electrical measurements at the single-particle level. A plasmonic double nanohole (DNH) nanopore sensor, consisting of an inverted snowman–shaped aperture patterned into a gold film and aligned with a nanoscopic pore in a silicon nitride membrane, enables optical trapping of individual particles above the nanopore while concurrent ionic current measurements are acquired. This multimodal platform was applied to AAVs containing single-stranded and self-complementary genomes, which have nearly identical total mass but potentially distinct internal DNA organization, enabling discrimination of fractionally filled subpopulations using combined optical–electrical signatures and unsupervised learning. Building on this approach, the platform was extended to include alternating-current (AC) electrical interrogation using both a conventional wire electrode and an integrated nanofabricated ring electrode. Frequency-dependent responses measured across these electrode configurations for silica and gold nanoparticles, proteins, and ultimately AAVs were shown to separate overlapping populations in multidimensional feature space, indicating improved separability between closely related AAV genome load states. iii Overall, this work demonstrates that combined optical and frequency-resolved electrical measurements of individual AAV particles provide a promising, label-free pathway for improved discrimination of genome load states in heterogeneous samples. By extending plasmonic nanopore sensing beyond conventional DC measurements, this dissertation establishes a foundation for high-dimensional, single-particle analytical metrology, with future work focused on leveraging the demonstrated separability of single-particle signatures for robust clustering and classification in heterogeneous AAV samples.
Keywords
plasmonic nanopore, adeno-associated virus, gene therapy, optical-electrical detection, nanopores, nanoparticles, AC modulation
Disciplines
Biomedical Engineering and Bioengineering
License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Renkes, Scott, "Plasmonic Nanopore Sensing for the Characterization of Viruses and Biomolecules" (2025). Bioengineering Dissertations. 210.
https://mavmatrix.uta.edu/bioengineering_dissertations/210