Label-free detection of HPV mRNA with an artificial chaperone-enhanced MNAzyme (ACEzyme)-based electrochemical sensor

Nucleic acid biosensors for point-of-care (POC) diagnostic applications are highly desirable. The ability to detect DNA and RNA in a simple, rapid, affordable and portable format leads to a range of important applications for early screening in the field of disease monitoring and management. Herein, we report the development of an isothermal, label-free electrochemical biosensor that was designed on the basis of target-driven MNAzyme cleavage activity. Hybridization with HPV mRNA, a model nucleic acid target, activated MNAzyme and initiated the cleavage of immobilized hairpin substrates, leading to changes in the electrochemical signal. Under optimal conditions, a detection limit of 2.6 pM was obtained with an incubation time of 60 min. Furthermore, an artificial chaperone-enhanced MNAzyme (ACEzyme) system was integrated to an electrochemical biosensor for the first time. The analytical performance of the biosensor was enhanced, and the detection time was significantly reduced by the addition of PLL-g-Dex, which exhibits nucleic acid chaperone-like activity. A detection limit of 0.88 pM was obtained with a threefold decrease in incubation time without prior amplification. The proposed biosensing platform shows the advantages of simple fabrication and operation, good selectivity in the presence of single-base mismatch, and excellent versatility in a complex mixture of total RNA. We believe that this isothermal, label-free, and protein-free nucleic acid analysis platform could provide foundations for the further development of a universal nucleic acid biosensing platform for clinical application.