Towards point-of-care tests: Comparative study of different antibody densities for plasmonic surfaces to achieve custom IL-18 biosensors

This work focuses on molecular recognition layer (MRL) characteristics because, in carbodiimide-mediated receptor immobilization on carboxyl-terminated self-assembled monolayers, the antibody concentration is crucial, as it can modulate surface crowding, probe orientation, and the fraction of binding-competent sites. More specifically, how changes in antibody concentration during the functionalization process influence the resulting MRL and whether these features translate into measurable differences in surface plasmon resonance (SPR) biosensor performance of disposable SPR-plastic optical fibre (POF) biosensors for IL-18 detection were investigated. With this aim, gold nanofilms on planar substrates and SPR plastic optical fibre (POF) platforms were functionalized and studied for IL-18 detection. Using planar chips, MRLs were first characterized by fluorescence microscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS), which indicated a marked dependence of the exposed antibody domains on the loading conditions. Finally, the impact on binding sensitivity for IL-18 detection was assessed using SPR-POF probes as a proof-of-concept. Two SPR-POF biosensors, obtained using different antibody concentrations, are evaluated for selectivity and sensitivity. Both produced negligible responses to interferents and quantified samples in simulated diluted serum. However, different dose-response curves changing the antibody concentration are achieved, showing that at low antibody concentration delivered substantially enhanced low-concentration sensitivity (0.25 nm/pM vs 0.053 nm/pM) and a lower detection limit (1.06 pM vs 10.8 pM), with an operating range shifted toward lower concentrations (1.06–25 pM vs 10.8–250 pM). Overall, this work links surface-chemistry evidence to functional SPR outcomes, showing that, for the disposable IL-18 SPR-POF biosensors, Ab concentration during carbodiimide coupling is a practical parameter to tune sensing performance, with the potential to reduce the cost per chip and improve SPR-biosensor performance simultaneously, characteristics useful for point-of-care tests. • Ab concentration in carbodiimide-mediated SAMs coupling reshapes the MRL conformation • Functionalized gold-nanofilm surfaces were developed and studied • Lower and higher anti-IL-18 Ab loading favoured different analyte range detection • SPR probes with different MRLs are investigated • A strategy to tune the SPR biosensor dynamic range is suggested