PolyA-mediated interfacial assembly of probes for label-free electrochemical detection of trastuzumab

PolyA-mediated spatial regulation enables modification-free probe assembly on gold electrodes. • An optimized AAP30 spacer promotes an ordered interfacial probe arrangement for efficient antibody recognition. • The engineered interface integrates label-free electrochemical sensing with reduced cellular adhesion. • The platform supports sensitive trastuzumab detection in serum and advances therapeutic antibody analysis. The analytical performance of heterogeneous biosensors is critically governed by the interfacial arrangement of recognition probes, as non-directional immobilization often limits binding-site exposure and hinders the recognition of bulky biomacromolecules. Herein, an interfacial engineering strategy based on the nanoscale spatial regulation of modification-free probes was developed to construct a label-free electrochemical biosensor for the detection of therapeutic antibodies. Using a polyA-aptamer probe (AAP) composed of the antibody-specific aptamer and the terminal polyA segment, we achieved the oriented self-assembly of probes onto gold electrodes through sequence-dependent nucleobase‑gold affinity, eliminating the need for complex chemical coupling. By adjusting the length of the polyA segment, the interfacial organization of the probes was systematically regulated. Results demonstrated that an optimized AAP30 probe facilitated a transition from an overcrowded, disordered monolayer to a well-spaced, near-upright architecture. This rational interfacial arrangement provides sufficient steric freedom for probe reconfiguration, thereby enhancing target accessibility and epitope recognition. Furthermore, the highly hydrated, polyanionic DNA layer contributed to improved surface wettability and reduced cellular adhesion, supporting the applicability of the biosensor in biological matrices. Using trastuzumab as a model therapeutic antibody, the proposed platform exhibited sensitive, label-free detection with a wide linear range from 10 ng/mL to 100 μg/mL and a limit of detection (LOD) of 5.05 ng/mL. Overall, this polyA-mediated spatial regulation strategy offers a practical and adaptable platform for designing label-free electrochemical biosensors for therapeutic drug monitoring in complex biological samples. Download: Download high-res image (104KB) Download: Download full-size image