Proof-of-Concept Study for Rapid Detection of Zika Viral RNA Via DNA Biosensor

Zika virus is a flavivirus that causes disease in humans. Zika infection has been linked to severe medical conditions such as Guillain-Barre syndrome in adults and microcephaly in newborns of mothers infected during pregnancy. The possibility of severe consequences has led to the need for new diagnostic techniques to replace the current inefficient and time consuming methods. We conducted a proof-of-concept study to demonstrate that a selective DNA-biosensor with peroxidase activity is able to specifically detect DNA molecules with a Zika virus-specific sequence (zDNA). Our approach utilizes a G-quadruplex hemin binding DNA aptamer, an effective signaling mechanism that forms a DNAzyme with peroxidase activity able to catalyze the oxidation of tetramethylbenzidine (TMB). The enzymatic activity of the DNAzyme, termed Proxzyme, is activated only in the presence of zDNA through a proximity dependent DNAzyme formation mechanism. In a series of experiments we optimized (1) reaction buffer composition, (2) length of spacer sequence, and (3) DNAzyme type. The developed zProxyzyme is proven to selectively detect zDNA in the nano-molar range in under 16 minutes colormentrically. The biosensor shows promising selectivity and will be paired with an on-site diagnostic device previously developed by our lab that utilizes alternating current electrokinetic (ACEK) based capacitance sensing to decrease the limit of detection.