HCCO Nanoreactor-Integrated Electrochemical Paper-Based Analytical Device for Anti-EBNA1 IgA Detection toward Nasopharyngeal Carcinoma Screening

Abstract

Serological detection of anti-Epstein–Barr virus nuclear antigen 1 immunoglobulin A (anti-EBNA1 IgA) is clinically relevant to nasopharyngeal carcinoma screening, but conventional immunoassays rely on centralized instrumentation and multistep procedures. Here, we developed a disposable origami electrochemical paper-based analytical device integrating hollow C@CuO–CeO₂ nanoreactors (HCCO-ePAD) for label-free anti-EBNA1 IgA detection. HCCO was designed as a multifunctional sensing interface in which the hollow carbon framework supports charge and mass transport, Cu-containing domains provide an intrinsic cathodic redox response, and surface carboxyl functionalization enables covalent EBNA1 immobilization. Binding of anti-EBNA1 IgA to surface-bound EBNA1 formed a poorly conductive immunocomplex that restricted interfacial electron transfer, producing a concentration-dependent decrease in the differential pulse voltammetric current. Under optimized conditions, the HCCO-ePAD showed a linear response from 0.1 to 500 ng mL^–1, with a detection limit of 0.029 ng mL^–1, together with good selectivity, repeatability, reproducibility, and storage stability. Preliminary evaluation using serum samples from 15 patients with nasopharyngeal carcinoma and 15 healthy controls yielded an area under the receiver operating characteristic curve of 0.969 (95% confidence interval, 0.918–1.000). The HCCO-ePAD results were further compared with those obtained using a commercial enzyme-linked immunosorbent assay (ELISA). This work demonstrates an intrinsic-redox nanoreactor interface that integrates electrochemical transduction and bioreceptor immobilization within a disposable origami paper platform for decentralized serological analysis.