Detection of HPV 16 and 18 L1 genes by a nucleic acid amplification-free electrochemical biosensor powered by CRISPR/Cas9

Abstract

Cervical cancer, closely linked to Human Papillomavirus (HPV) infection, remains a significant health threat for women worldwide. Conventional HPV detection methods, such as reverse transcription polymerase chain reaction (RT-PCR), rely on nucleic acid amplification (NAA), which can be costly and time-consuming. This study introduces an NAA-free electrochemical Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based biosensor designed to detect HPV 16 and HPV 18 L1 genes simultaneously. The system utilizes a Cas9-single guided RNA complex to initiate a selective cleavage reaction, releasing Methylene blue or Ferrocene-labeled fragments correlate to L1 gene concentrations. These fragments then interact with modified gold electrodes immobilized with a complementary probe, allowing precise electrochemical signal measurement during hybridization. The biosensor offers a wide detection range from 1 fM to 10 nM, with detection limits as low as 0.4 fM for HPV 16 L1 and 0.51 fM for HPV 18 L1, providing a sensitive and efficient solution for L1 gene detection. Additionally, its specificity and sensitivity closely match RT-PCR results in clinical testing, highlighting its potential for molecular diagnostics and point-of-care applications.