CRISPR/Cas14a integrated with DNA walker based on magnetic self-assembly for human papillomavirus type 16 oncoprotein E7 ultrasensitive detection

Abstract

To enhance the biomarker diagnostics sensitivity and selectivity of human papillomavirus type 16 oncoprotein E7 (HPV16 E7) in serum, a label/enzyme-free electrochemical detection platform was developed. This platform featured a type of "Super-turn-off" nanobiosensor monitored through differential pulse voltammetry (DPV). It integrated the magnetic self-assembly property of the α-Fe₂O₃/Fe₃O₄@Au/Sub/BSA signal transport nano-medium with the high specificity of CRISPR/Cas14a and the amplification capability of the bipedal walker (DNA walker composed of two ssDNA strands), resulting in the enhanced specificity and anti-interference performance while remaining stable at 4 °C for over 30 days. The results demonstrated that the combination of walker and CRISPR yielded superior sensitivity and analytical capability compared with using either technology alone, achieving a detection limit of 67.17 fg mL⁻¹, a quantification limit of 0.22 pg mL⁻¹, and serum sample recovery rates of 98.46%–115.78%. Additionally, this platform was applied to detect serum and tissue samples from mouse models at various stages of cervical cancer, significantly improving the accuracy and effectiveness of early diagnosis and prognostic evaluation. This novel approach held promise as an efficient tool for point-of-care clinical detection of HPV16 E7, potentially reducing cervical cancer mortality.