Single-Tube Multiplex Detection of High-Risk HPV Subtypes Utilizing Lanthanide Nanoprobes

The detection of human papillomavirus (HPV) and its subtypes is of great significance, as the high expression of multiple high-risk subtypes is often associated with various deadly cancers. The golden standard polymerase chain reaction (PCR) has achieved remarkable success in HPV detection, saving many lives over the past few decades. Despite excellent analytical merits, PCR sometimes faces two challenges in the multiplex detection of HPV subtypes: (1) the commercial fluorescent dyes in a single-tube could occasionally be subject to the spectral overlapping interference; (2) the amplification process may introduce contaminations and false positive signals. Herein, the detection of six high-risk HPV subtypes (HPV-16, HPV-18, HPV-31, HPV-39, HPV-56, and HPV-58) in a single-tube was achieved, utilizing self-synthesized lanthanide nanoprobes (NaTbF₄, NaHoF₄, NaEuF₄, NaPrF₄, NaYF₄, and NaTmF₄). These lanthanide nanoprobes feature a low biological background and high sensitivity due to their high intrinsic metal content, enabling the mass spectrometry-based sensitive detection. The detection limits of as low as 0.07 pM were achieved, eliminating the need for the additional nucleic acid amplification. Furthermore, the proposed method exhibited high accuracy and precision in clinical serum samples across all tested HPV subtypes.