Human papilloma virus DNA detection in clinical samples using fluorogenic probes based on oligonucleotide gated nanoporous anodic alumina films.

In this work, fluorogenic probes based on oligonucleotide capped nanoporous anodic alumina films are developed for specific and sensitive detection of human papilloma virus (HPV) DNA. The probe consists of anodic alumina nanoporous films loaded with the fluorophore rhodamine B and capped with oligonucleotides bearing specific base sequences complementary to genetic material of different high-risk (hr) HPV types. Synthesis protocol is optimized for scale up production of sensors with high reproducibility. Sensor´s surfaces are characterized by scanning electron microscopy (HR-FESEM) and atomic force microscopy (AFM) and their atomic composition is determined by energy dispersive X-ray spectroscopy (EDXS). Oligonucleotide molecules onto nanoporous films block the pores and avoid diffusion of rhodamine to the liquid phase. Pore opening is produced when specific DNA of HPV is present in the medium, resulting in rhodamine B delivery, that is detected by fluorescence measurements. The sensing assay is optimized for reliable fluorescence signal reading. Nine different sensors are synthesized for specific detection of 14 different hr-HPV types in clinical samples with very high sensitivity (100%) and high selectivity (93-100%), allowing rapid screening of virus infections with very high negative predictive values (100%). Sensors and sensing assay developed in this work allow multiplexed analysis of clinical samples resulting in faster screening which can be carried out by non-specialized personnel. This article is protected by copyright. All rights reserved.