Electrochemical biosensors represent mature platforms for point-of-need analysis due to their high sensitivity, intrinsic selectivity, low cost, and facile miniaturization. In the last decade, nanomaterials have become integral to advanced biosensor architectures, acting as high-surface-area supports, electron-transfer mediators, and signal-amplifying elements. This review critically examines the most represented nanomaterial classes in mature electrochemical biosensors—carbon nanostructures, gold nanoparticles, and iron-based magnetic nanoparticles—highlighting how morphology, electronic structure, and surface chemistry influence key performance metrics such as limit of detection, linear range, and assay time. Despite a strong technology push and numerous proof-of-concept demonstrations, the translation of nanomaterial-enabled electrochemical biosensors into commercial devices remains limited. This gap arises from the intrinsic physicochemical complexity of nanomaterials, which hampers standardization, reproducibility, and long-term safety assessment. Accordingly, this review integrates performance analysis with a systematic overview of the European regulatory framework, including the Medical Device Regulation (MDR) (EU) 2017/745, the In Vitro Diagnostic Regulation (IVR) (EU) 2017/746, EFSA guidance for food and water applications, and relevant ISO standards, outlining key translational bottlenecks and design principles for deployable biosensing technologies.

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.