Ultrasensitive detection of HPV-associated lower genital tract lesions using a NIR-excited luminescent assay with lanthanide-doped upconversion nanoprobes and enzymatic digestion protection.

Persistent high-risk human papillomavirus (HPV) infection is a major cause of cervical carcinogenesis, yet current diagnostic methods - particularly colposcopy with nonspecific dyes - lack the precision needed for early-stage precancerous lesion detection. To address this gap, we developed a luminescent nanoprobe platform based on lanthanide-doped upconversion nanocrystals for HPV E7 oncoprotein detection, featuring three key advancements: (1) The nanoprobes exploit long luminescence lifetimes and near-infrared (NIR) emission to minimize background autofluorescence and enhance tissue penetration, while epitope -specific binding to E7 antigens ensures high target selectivity. (2) A homogeneous detection scheme, defined as the final solution-phase fluorescence reading obtained after proteolytic digestion, wherein proteolysis triggers cell lysis and probe release, enables precise fluorescent signal acquisition with a detecting limit of 5 cells/well, while preserving probe integrity for potential reuse. (3) A multichannel targeted imaging strategy suppresses tissue scattering artifacts, significantly improving early-stage lesion detection rates (p < 0.001). This work establishes a novel HPV diagnosis platform with enhanced sensitivity and clinical detection performance.