High-sensitivity electrochemical detection of HPV DNA via enzyme-amplified target-induced hairpin opening on a thermally controlled paper-based digital microfluidic platform.

Human papillomavirus, a leading cause of cervical cancer, poses a global health threat. Rapid and accurate point-of-care testing (POCT) is crucial for early detection and disease control. Here, we present an electrochemical sensor on a thermally controllable paper-based digital microfluidic (e-pDMF) device for detecting human papillomavirus type 16 DNA (HPV16 DNA) via target-induced hairpin opening and enzyme-assisted signal amplification strategy. The e-pDMF device operates in transport and thermal modes to enable programmable droplet manipulation and precise temperature control via an integrated thermal platform capable of heating to 125 °C. In this study, the platform maintained a 37.5 ± 0.14 °C isothermal state with a 0.09 °C/s ramp rate to support efficient Exonuclease III activity during signal amplification. The integrated system facilitates multiple processes, from sample loading and mixing to signal amplification and electrochemical detection, and completes the whole assay within 75 min. The products from the enzyme-amplified target-induced hairpin opening process can be captured on the probe-immobilized electrode. Differential pulse voltammetry measures the current decrease caused by electron transfer of redox indicator, indicating the presence of target HPV16 DNA. The sensing platform demonstrated a detection range from 1 fM to 10 pM (R = 0.9968), with an experimental detection limit of 1 fM. It showed high specificity for HPV16 DNA, with no cross-reactivity to mismatched sequences or related genital tract pathogens. The platform was validated using clinical cervical swab samples, showing 100 % agreement with PCR results. This portable and automated e-pDMF device shows great potential as an alternative POCT tool for DNA diagnosis.