To overcome early cancer detection challenges, diagnostic tools enabling more sensitive, rapid, and noninvasive detection are necessary. An attractive cancer target for diagnostic blood tests is human Ecto-NOX disulfide–thiol exchanger 2 (ENOX2), expressed in most human cancer types and regularly shed into blood sera. Here, we developed an electrochemical DNA-based (E-DNA) biosensor that rapidly detects physiologically relevant levels of ENOX2. To identify ENOX2-binding aptamers that could potentially be used in a biosensor, recombinantly expressed ENOX2 was used as a binding target in an oligonucleotide library pull-down that generated a highly enriched ENOX2-binding aptamer. This candidate aptamer sensitively bound ENOX2 via gel mobility shift assays. To enable this aptamer to function in an ENOX2 E-DNA biosensor, the aptamer sequence was modified to adopt two conformations, one capable of ENOX2 binding, and one with disrupted ENOX2 binding. Upon ENOX2 introduction, a conformational shift to the ENOX2 binding state resulted in changed dynamics of a redox reporter molecule, which generated a rapid, significant, and target-specific electrical current readout change. ENOX2 biosensor sensitivity was at or below the diagnostic range. The ENOX2 E-DNA biosensor design presented here may enable the development of more sensitive, rapid, diagnostic tools for early cancer detection.

Carrion’s disease, caused by infection with the bacterium Bartonella bacilliformis (B. bacilliformis), is effectively treated with antibiotics, but reaches fatality rates of ~90% if untreated. Current diagnostic methods are limited, insufficiently sensitive, or require laboratory technology unavailable in endemic areas. Electrochemical aptamer-based (E-AB) biosensors provide a potential solution for this unmet need, as these biosensors are portable, sensitive, and can rapidly report the detection of small molecule targets. Here, we developed an E-AB biosensor to detect Pap31, a biomarker of Carrion’s disease and an outer membrane protein in B. bacilliformis. We identified an aptamer with Pap31-specific binding affinity using a magnetic pull-down assay with magnetic bead-bound Pap31 and an aptamer library followed by electrophoretic mobility shift assays. We incorporated the Pap31-binding aptamer into a DNA oligonucleotide that changes conformation upon binding Pap31. The resultant Pap31 E-AB biosensor produced a rapid, significant, and target-specific electrical current readout in the buffer, demonstrating an apparent K_D of 0.95 nM with a limit of detection of 0.1 nM, and no significant signal change when challenged with off-target proteins. This proof-of-concept Pap31 biosensor design is a first step toward the development of more rapid, sensitive, and portable diagnostic tools for detecting Carrion’s disease.