Development of a highly sensitive, label-free electrochemical immunosensor for p16INK4a detection: a step toward early cervical cancer diagnosis†

Cervical cancer screening is a key public health approach for the secondary prevention of cervical cancer, particularly in resource-limited countries. This study presents the development of a highly sensitive electrochemical immunosensor for the detection of p16INK4a, a protein overexpressed in cervical cancer. The immunosensor was constructed using a layer-by-layer hybrid film of gold nanoparticles (AuNPs) and reduced graphene oxide (rGO) co-deposited on glassy carbon electrodes (GCEs), which improved electrical conductivity and effective surface area for immobilizing the capture antibody. Anti-p16INK4a monoclonal antibodies were immobilized on the modified electrode surface through a cystamine-glutaraldehyde crosslinking method. Square wave voltammetry (SWV) was employed for p16INK4a detection, achieving a limit of detection (LOD) of 167 fg mL⁻¹ and a linear detection range of 500 fg mL⁻¹ to 100 ng mL⁻¹ under optimal conditions. The proposed immunosensor exhibits outstanding selectivity, storage stability, reproducibility, and regeneration capabilities. The application of the immunosensor to clinical samples (positive: n = 10; healthy: n = 5) demonstrated high accuracy, with results aligning well with those obtained from the enzyme-linked immunosorbent assay (ELISA). These findings indicated that the proposed sensing platform can be utilized for the early diagnosis of cervical cancer.