Development of an Electrochemical, DNA‐Based Biosensor to the Cancer Biomarker ENOX2

Abstract

One challenge to the identification of cancerous malignancies is the ability to detect cancer‐specific antigens in a rapid, reliable, and accessible manner. Human Ecto‐NOX Disulfide‐Thiol Exchanger 2 (ENOX2) is a cell‐surface metalocatalyst frequently expressed on virtually all major human cancer types and is critical for cell maturation. Importantly, ENOX2 regularly sheds into the sera, making it an attractive target for diagnostic blood testing. As a strategy for diagnostics focused on utilizing these shed ENOX‐2 particles, we here demonstrate an electrochemical DNA‐based (E‐DNA) biosensor directed against ENOX‐2 in solution. To do so, we first exploited a modified SELEX (systematic evolution of ligands by exponential enrichment) protocol to generate aptamers against ENOX2 recombinantly expressed in E. coli. A pool of candidate aptamers was identified by high‐throughput sequencing, and gel mobility shift assays were used to characterize the best candidate aptamers and classified those with the highest affinity for ENOX2. The leading aptamer sequence was used as the core of our E‐DNA biosensor, wherein changes in DNA conformation upon target binding result in changed dynamics of an appended redox reporter molecule, ultimately generating a rapid and quantitative change in the current of the electrochemical response when interrogated via voltametric methods such as square‐wave voltammetry. Our sensor displays robust response to ENOX2 in solution, and may allow accelerated, dose‐responsive readout for the presence of ENOX2 in both buffer and blood serum.