FFF as a microfluidic platform for the streamlined optimization of ready-to-use nanozyme-labelled probes to enable robust and ultra-sensitive chemiluminescent bioassays

The replacement of enzyme labels such as horseradish peroxidase (HRP) with noble metal nanoparticles represents a promising approach in biosensing. Ultrasmall platinum nanoparticles (PtNPs) exhibit remarkable catalytic activity, chemical stability, and low production costs, making them attractive nanozyme candidates also in the field of highly sensitive chemiluminescence (CL) detection. However, their CL performances strongly depend on particle size, shape, composition, and concentration, often resulting in limited reproducibility and robustness. These physicochemical parameters also affect biomolecular labeling efficiency, including conjugation with proteins and antibodies, which is critical for consistent biosensor performance. To enable the development of robust biosensors integrating nanozymes as active sensing elements, rigorous nanoparticle quality control and efficient isolation of their bioconjugates are key steps. In this work, we report the first application of hollow-fiber flow field-flow fractionation (HF5) for the characterization and isolation of bioconjugated ultrasmall PtNPs designed for CL bioassays. Shape-controlled pyramidal PtNPs were synthesized in aqueous media via a rapid, scalable, and surfactant-free route exploiting sodium citrate as a shape-directing agent. PtNPs were characterized to define optimal nanoparticle-to-protein ratios, ensuring high conjugate stability. HF5 was then employed to selectively isolate standardized conjugated PtNPs, which were directly employed for CL signal generation. Finally, a proof-of-concept study demonstrated the use of conjugated IgG-PtNPs isolated through HF5 for the quantitative detection of a primary antibody in a paper-based CL bioassay, achieving a limit of detection suitable for diagnostic applications. Thus, HF5-mediated streamlining of probe synthesis and isolation offered a very promising answer to unmet needs in biosensor development.