Novel electrochemical real-time polymerase chain reaction utilizing silver ion aptamer-modified primer

Abstract

We herein describe a novel electrochemical real-time polymerase chain reaction (ERT-PCR) utilizing silver ion ($Ag^+$) aptamer-modified primer. In this work, we designed the primer to be extended by $Ag^+$ aptamer at the 5’ end, which would initially form a hairpin structure entrapping $Ag^+$. The $Ag^+$ in this bound state cannot actively participate in the redox reaction due to the low diffusivity, resulting in the low electrochemical signal. In the presence of target DNA, however, the $Ag^+$ is released from the aptamer as the opposite DNA strand is synthesized or as the $Ag^+$ aptamer-modified primer anneals to the extended template during the PCR process. Since the released free $Ag^+$ has much higher diffusivity than the bound one, the $Ag^+$-mediated electrochemical signal would increase in accordance with the number of PCR cycles. Based on this simple design principle, we successfully detected the Enterococcus faecium (E. faecium) genomic DNA (gDNA) down to $3.085 × 10^2 copies/\mul$ with excellent selectivity against non-target DNAs. This method has a great potential of serving as a key platform technology to advance an electrochemical genetic diagnostic system for a point-of-care testing (POCT) purpose.