Highly sensitive detection of adeno-associated virus serotype 9 enabled by a nanobody-functionalized multiphase TiO2 photoelectrochemical immunosensor.

The reliable detection and quantification of adeno-associated virus serotype 9 (AAV9) is crucial for gene therapy development and quality control. This work presents a significant advancement by developing a novel, nanobody-based photoelectrochemical (PEC) immunosensor for the highly sensitive and specific detection of AAV9. The core is a laser-synthesized multiphase TiO2 heterojunction photoanode fabricated directly on a titanium substrate. This one-step, mask-free laser processing technique ensures excellent material consistency and superior photocurrent response, overcoming the batch-to-batch variability associated with traditional electrode modification methods. The sensing interface is further engineered by immobilizing a high-affinity anti-AAV9 nanobody, which offers minimal steric hindrance and enhanced epitope accessibility compared to conventional antibodies. The synergistic integration of a high-performance multiphase photoactive material with a biological recognition element, resulting in a satisfactory sensing platform. The fabricated immunosensor demonstrates a wide linear detection range from 5 × 107 to 1 × 1010 vg/mL (vg/mL: viral genomes per milliliter) for AAV9, with a remarkably low calculated detection limit (LOD) of 1 × 107.2 vg/mL and limit of quantitation (LOQ) of 3 × 107.2 vg/mL. To our knowledge, this represents the first successful application of an AAV9-specific nanobody within a PEC biosensing platform. This study not only provides a powerful tool for virological analysis but also establishes a versatile framework that can be adapted for detecting other viral targets by exchanging the nanobody probe, showcasing broad potential in biomedical diagnostics.