Renewable nanocrystal capture interfaces for greener paper-based immunoassays: Comparative design rules from interfacial metrology to colorimetric prototypes

Abstract: Renewable polysaccharides can serve as both the substrate and the functional capture interface in point-of-need diagnostics, enabling water-first fabrication and reducing reliance on persistent petrochemical components. Here, we present a comparative framework that benchmarks polysaccharide nanocrystals as antibody supports for paper-based immunoassays by integrating physicochemical characterization, orthogonal interfacial metrology, and prototype translation. Specifically, we compare cellulose nanocrystals (CNCs), chitin nanocrystals (ChNCs), and CNC/chitosan (CNC/Ch) composites as supports for antibodies targeting human CD66e (CEACAM5), an established biomarker for epithelial cancers. Nanocrystal platforms were characterized to connect molecular composition and electrostatics to probe presentation and interfacial behavior (morphology, charge, and accessible functional groups). Antibodies were immobilized using carbodiimide/N-hydroxysuccinimide coupling under aqueous conditions, and antigen recognition was quantified in real time using quartz crystal microbalance with dissipation monitoring (QCM-D) and multiparameter surface plasmon resonance (MP-SPR) across a stepwise antigen concentration series to evaluate saturation behavior and concentration-dependent responses. The paired QCM-D/MP-SPR readout is central: it enables mechanistic interpretation of soft biopolymer films where hydration, viscoelasticity, and interfacial restructuring can dominate “apparent mass” signatures, potentially confounding comparisons if a single technique is used. Across platforms, CNC/Ch composites deliver superior antibody immobilization efficiency and stronger antigen-binding responses than CNC or ChNC supports, consistent with synergistic cationic chitosan–high-surface-area CNC interfaces that increase effective capture density while maintaining practical processing. To demonstrate translational relevance, these renewable capture layers are deployed on additive-free paper and paired with gold-nanoparticle labels in a simple colorimetric prototype compatible with replicate testing and short assay times. Building on our broader detection portfolio (including rapid dipstick-style assays on cellulose-based interfaces), this work provides green-chemistry-aligned design rules for renewable immunoassays that are scalable, fieldable, and grounded in interfacial structure–function relationships.