Single Particle Adsorption and Response Quantification for Functional AAVx Titer and Dose Analysis

Accurate quantification of functional adeno-associated virus (AAV) dose remains a critical limitation in gene therapy, where titers are defined using ensemble measurements of viral genomes or capsid concentration that average across heterogeneous populations and weakly predict transduction. We present single-particle adsorption and response quantification (SPARQ), a diffraction-limited imaging platform for quantifying AAVx vectors, regardless of serotype, at single-nanoparticle and single-cell resolution. SPARQ immobilizes and resolves individual AAVx, enabling concurrent measurement of capsid-associated and genome-associated signals before cell exposure. Across multiple serotypes, including AAV2, AAV5, and AAVrh74, SPARQ identifies full-capsid fractions, consistent with bulk measurements obtained by ddPCR/ELISA, DLS/UV-Vis, charge detection, and mass photometry, while reporting narrower distributions than ensemble-derived values and revealing systematic discrepancies in bulk-derived titers arising from population averaging. Surface immobilization is achieved by charge-dependent AAVx-surface interactions, as supported by multiphysics simulations, which capture serotype-dependent adsorption kinetics governed by capsid electrostatics for AAV2, AAV5, AAV6, and AAV9. Furthermore, SPARQ directly measures transduction as a function of particle number and reveals a threshold response. Transduction efficiency increased from ∼29% (S.D ± 3.31%) at ∼18 (S.D ± 7.55) AAVs per cell to ∼66% (S.D ± 1.66%) at ∼42 (S.D ± 9.69) AAVs per cell, which is comparable with conventional in vitro assay requiring 10 ⁵ AAVs/cell. Cumulative fluorescence measurements across large micro-patterned arrays of single cells recapitulate bulk-like high-throughput scaling, while SPARQ’s single-nanoparticle resolution reveals functional heterogeneity that is masked by population averaged assays. SPARQ provides a particle-resolved framework for functional AAVx titration and quantitative characterization, enabling the development of quality control and dosing strategies in gene therapy manufacturing.