Methylene blue-modified hexagonal boron nitride nanostructured immunosensor for early detection of HPV16 E6 oncoprotein.

Human papillomavirus type 16 E6 oncoprotein is a clinically significant biomarker for cervical cancer because its persistent expression drives malignant transformation. To address the need for ultrasensitive detection of oncogenic HPV16 E6 protein, we engineered an electrochemical immunosensor based on methylene blue-functionalized hexagonal boron nitride (BN-MB) nanosheets deposited on screen-printed carbon electrodes. In this architectonic design, exfoliated h-BN provides a high-surface-area support, while the π-π-stacked cationic dye methylene blue acts as an internal redox mediator, converting intrinsically insulating h-BN into an electroactive matrix. Protein G was immobilized to orient monoclonal anti-E6 antibodies in a tail-on configuration, thereby enhancing antigen accessibility. Under optimized conditions, square-wave voltammetry showed a wide linear range from 1 fg mL⁻¹ to 1 ng mL⁻¹, with an ultralow limit of detection of 0.80 fg mL⁻¹ using the 3σ/m method and a limit of quantification of 2.67 fg mL⁻¹. Zeta potential measurements and FTIR analysis confirmed strong electrostatic and π-π interactions between MB and h-BN, while MB loading was determined by UV-vis absorbance. The immunosensor exhibited excellent selectivity against a panel of interferents, including E7 oncoprotein, SCCA, CEA, and CA-125, high stability with approximately 87% signal retention after 30 days at 4 °C, and good reproducibility with a relative standard deviation of 3.20%. Analysis of E6-spiked human serum samples and samples collected from seven HPV-positive patients showed generally good agreement with commercial ELISA, while some variability was observed among individual clinical samples. Overall, the BN-MB immunosensor demonstrates an innovative material platform for point-of-care screening of cervical cancer biomarkers.