Abstract

In the face of escalating global concerns regarding nanoplastic pollution, we report a homogeneous immunoassay platform that integrates nanobody engineering with NanoLuciferase Binary Technology, establishing a paradigm shift in sub-50 nm nanoplastics quantification. This breakthrough system utilizes a polystyrene-targeting nanobody (B2) we previously screened, which is genetically fused to split NanoLuciferase subunits (SmBiT/LgBiT). Through the reconstitution of luciferase subunits, it enables real-time luminescent signal amplification upon the binding of nanoparticles. Through systematic optimization, this assay achieves non-destructive quantification of 20 nm polystyrene nanoparticles within 5–10 min without sample pretreatment, featuring a detection limit of 9.1 μg/mL and a limit of quantification of 19.9 μg/mL. Its robustness and applicability were confirmed through spike-recovery tests in five types of real environmental matrices, yielding recoveries of 86.52–146.44 % and coefficients of variation of 9.1–18.2 %. The practical utility of this method was further verified using commercial polystyrene products. This innovative strategy disrupts conventional detection methodologies by eliminating the need for costly instruments or professional operations, while enabling microvolume analysis (10–20 μL) down to 20 nm. As a versatile framework for nanoplastic detection, it paves the way for on-site environmental monitoring and biomedical research, addressing critical gaps in current nanoplastic quantification technologies and holding significant promise for mitigating ecological and health risks associated with nanoplastic contamination.