Improved serum biomarker detection via self-clickable CuO nanoparticle-coated Ag slides with oriented and stable antibody presentation on antifouling surfaces.

We propose an innovative approach to immobilize antibodies (Abs) on solid supports, addressing challenges in generating immunosensor arrays. This method involves a self-clickable CuO-nanoparticle-coated silver (CuO@Ag) surface for boronic acid (BA) functionalization, facilitating the irreversible immobilization of native Abs. The surface catalyzes azide-alkyne cycloaddition with azido-containing BA molecules without requiring an external Cu(I) ion source. Simultaneously, a metal-enhanced fluorescence effect amplifies the array detection signal, enabling ultrasensitive serum biomarker measurement. This process improves Ab orientation, maximizing antigen-binding site exposure for target protein interaction. The CuO@Ag glass slides show enhanced sensitivity, reduced nonspecific adsorption, and detection limits of 0.15, 0.07, 1.70, and 0.24 ng/mL for CRP, SAP, SAA, and PSA detection, respectively, which are below the diagnostic thresholds. When validated in a multiplex format with human serum samples, the assay demonstrated strong specificity and sensitivity in detecting key cancer biomarkers (CRP, SAP, SAA, AFP, CEA, and PSA). This CuO@Ag strategy for nonengineered Ab immobilization has broad implications for improving affinity-based protein detection assays.