Integrated microfluidic device for rapid and accurate point-of-care detection of high-risk HPV16 and HPV18

Abstract

Human papillomavirus (HPV), particularly high-risk genotypes HPV16 and HPV18, is a leading cause of cervical cancer, contributing to significant global morbidity and mortality, especially in low-resource settings. Current diagnostic methods, such as PCR and cytology-based Pap tests, are limited by high costs, complex infrastructure requirements, and variable sensitivity, hindering their accessibility in low- and middle-income countries. To address these challenges, an integrated microfluidic platform was developed for rapid, sensitive, and point-of-care (POC) detection of HPV16 and HPV18 DNA. This platform combines loop-mediated isothermal amplification (LAMP) with lateral flow assay (LFA) detection, enabling a streamlined “sample-in-answer-out” workflow. The cartridge integrates nucleic acid (NA) extraction, isothermal amplification at 65 °C, and visual detection within 45 minutes, utilizing a lysis buffer optimized for sample preparation and genotype-specific primers targeting the E6 genes of HPV16 and HPV18. The microfluidic design leverages the Zweifach-Fung bifurcation principle for efficient NA separation, eliminating the need for centrifugation or external equipment. Analytical validation using spiked clinical samples demonstrated a limit of detection of 10 copies per µL, with T/C ratio-based quantification showing strong linearity (R² > 0.98) over a dynamic range of 10⁸ to 1 copy per µL. A clinical evaluation of 50 cervical swab samples yielded a sensitivity of 96.8% and a specificity of 100% compared to qPCR, with positive and negative predictive values of 100% and 94.7%, respectively. The platform's portability, low cost, and minimal user intervention make it an ideal solution for decentralized settings, offering a transformative approach to early HPV screening and cervical cancer prevention in resource-limited environments.