Nanocomposite-based all-in-one electrode coating towards affinity-based electrochemical biosensing platform

The commercialization of affinity-based electrochemical point-of-care devices remain constrained by their reliance on external redox mediators and the longstanding challenge of simultaneously achieving high signal sensitivity and antifouling capability. Herein, we report a hierarchical nanocomposite-based all-in-one electrode coating that strategically integrates intrinsic redox activity, antifouling capability, and high conductivity into a unified, drop-castable interface. The nanocomposite comprises of redox-active ferrocene-functionalized Ce-UiO-66-NH₂ metal-organic framework (Ce-MOF-Fc) and highly conductive polyaniline nanowires (PANI NWs) embedded within glutaraldehyde-crosslinked bovine serum albumin (GB) matrix. The GB matrix suppresses nonspecific adsorption and retains 84% signal after extended exposure to fouling biofluids. Its recessed, open-pore morphology and percolated PANI NWs network enable efficient charge transport and amplifies the intrinsic redox signal of Ce-MOF-Fc. Furthermore, the nanocomposite supports facile covalent immobilization of amine-terminated biorecognition elements (e.g., aptamers, antibodies) via 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride/N-hydroxy succinimide (EDC/NHS) activation of bovine serum albumin (BSA) carboxyl groups. As a proof-of-concept, an epithelial cell adhesion molecule (EpCAM)-specific aptamer (Apt-NH₂) was designed and immobilized for label-free detection of M D Anderson-Metastatic Breast −231 (MDA-MB-231) breast cancer cells. The biosensor exhibited linearity from 5 to 800 cells/mL, a detection limit of 5 cells/mL, high reproducibility, and excellent specificity in simulated serum and urine. This modular and versatile platform offers a valuable blueprint for next-generation electrochemical affinity biosensors with broad applicability in real-world biomedical diagnostics.