Ring-Electrode AC Plasmonic Nanopore Sensing for DNA Load Characterization of Single Adeno-Associated Viruses

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Abstract
Reliable quality control of adeno-associated virus (AAV) vectors remains a major bottleneck in gene therapy manufacturing, particularly for resolving subtle differences in genome loading and conformation at the single-particle level. Existing approaches often struggle to distinguish AAV populations with similar mass and charge, such as capsids carrying self-complementary versus single-stranded DNA. Here, we introduce an AC plasmonic nanopore sensing framework for AAV9 characterization. Individual AAV capsids were optically trapped within a plasmonic double-nanohole nanopore and interrogated using multi-frequency AC pulse trains spanning 500 Hz to 100 kHz. To enhance sensitivity to localized particle–field interactions, a nanofabricated Ag/AgCl ring electrode was integrated concentrically with the plasmonic nanopore. Relative to a conventional wire electrode, the ring electrode produced broader and more robust analyte-dependent differences across multiple frequency-dependent parameters, enabling reliable discrimination of empty capsids (AAVempty) and genome-loaded capsids carrying either self-complementary (AAVscDNA) or single-stranded DNA (AAVssDNA), despite their near-identical genome mass. Concentration titration experiments further demonstrated that the extracted multivariate AC features remained largely concentration-independent over the tested range. Together, these results demonstrate that ring-electrode-enabled AC plasmonic nanopore sensing provides a multidimensional framework for resolving closely related AAV populations and advances plasmonic nanopores toward practical single-particle quality control of gene therapy vectors.
Funding Information
  • National Science Foundation (NSF CBET#2421778)
  • National Science Foundation (#2421779)
  • National Science Foundation (TI#2415309)
  • National Institutes of Health (NIH/NIGMS 1R01GM149949-01)

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