Development of a Microfluidic Point-of-Care Platform for HPV Detection Based on Helicase-Dependent Amplification

Human papillomavirus (HPV) is the most prevalent sexually transmitted infection worldwide and a leading cause of cervical cancer, accounting for over 300,000 deaths annually, primarily due to high-risk genotypes HPV-16 and HPV-18. Conventional molecular diagnostic methods, such as polymerase chain reaction (PCR), require expensive instrumentation and well-equipped laboratories, which limits their applicability in low-resource or decentralized settings. To address this challenge, the aim of this study was to develop a prototype point-of-care (POC) diagnostic platform based on helicase-dependent amplification (HDA) integrated into a microfluidic device for the specific detection of HPV-16 and HPV-18. The proposed POC platform comprises a disposable poly (methyl methacrylate) (PMMA) microfluidic device, a portable warming mat for isothermal amplification at 65 °C, and a compact electrophoresis chamber for fluorescence-based visualization using SYBR Safe dye, with an approximate total cost of $320 USD. Platform validation was performed on 33 samples, demonstrating amplification of target sequences in less than 60 min with only 20 µL of reaction volume, a limit of detection (LOD) of 15 copies (cp) per reaction, a sensitivity of 95.52%, and a specificity of 100%. This portable and scalable platform constitutes a cost-effective and reliable tool for the detection of HPV, supporting global health initiatives, including those driven by the World Health Organization (WHO), aimed at eliminating cervical cancer as a public health threat, as it can be implemented in decentralized or resource-limited settings.