Fluorescence biosensing for simultaneous detection of HPV16 and HPV18

Cervical, most anal, and most oropharyngeal cancers are caused by human papillomavirus (HPV), with HPV16 and HPV18 accounting for the majority of cases, necessitating simultaneous detection of multiple HPV types for identifying HPV infection, risk stratification, and screening for HPV-associated disease. Entropy-driven catalytic strand displacement reactions are DNA self-assembly amplification methods that do not require enzymes for signal amplification. Furthermore, their driving forces originate within the system, endowing them with high specificity, which provides a crucial advantage for simultaneous detection of several targets. Therefore, an analytical method was developed to amplify fluorescence signals via dual-toehold-mediated strand displacement, enabling simultaneous detection of HPV18 and HPV16. For both targets, the established method shows excellent linearity of the regression curve in the concentration range from 0.05 to 5 nM. The detection limits for HPV18 and HPV16 were 0.020 and 0.033 nM, respectively, which are highly favorable even for the detection of only a single HPV type. These results show that the developed enzyme-free assay has high sensitivity and specificity, making it a promising technique for rapid and cost-effective HPV16 and HPV18 screening.