Electrochemical DNA Biosensor to Detect Glycopeptidolipids of Nontuberculous Mycobacteria

Abstract

In many regions, nontuberculous Mycobacteria (NTM) pulmonary infections have outnumbered tuberculosis infections over the past 30 years, yet NTM infections are frequently misdiagnosed. One critical challenge is that the current “gold standard” for NTM diagnosis involves a weeks‐to‐month long microbiological culture‐based method. To provide a more efficient diagnostic method, we have developed a novel electrochemical DNA‐based (E‐DNA) biosensor to detect NTM. Our E‐DNA biosensor targets a mycobacterial glycopeptidolipid (GPL) found on the exterior cell wall of NTM, which demonstrates high exclusivity for NTM. To create the E‐DNA biosensor, we successfully generated a GPL‐specific single‐stranded DNA aptamer through a modified SELEX (systematic evolution of ligands by exponential enrichment) process followed by high throughput sequencing to quantify potential aptamer sequences. The putative aptamers were then synthesized and screened via fluorescence, with the best candidate aptamer displaying a dissociation constant in the low nanomolar range (KDapp = 0.73 nM). Computational analysis was used to predict secondary and tertiary structure of the aptamer, allowing optimization of that aptamer sequence to find the minimum binding domain and engineer in a two‐state folding mechanism. The aptamer will then be synthesized with a 5’ thiol linkage to facilitate attachment to a gold electrode along with an internal redox reporter (methylene blue). This will enable an electrochemical interrogation method wherein changes in the peak current in square wave voltametric analysis correlate to a conformational rearrangement of the aptamer upon binding to GPL. In future studies, this E‐DNA biosensor will be verified against NTM‐positive patient samples. Ultimately, this approach should provide a rapid, point‐of‐care diagnostic method that eliminates the current lengthy culturing requirements to shorten the duration between the onset of symptoms and correctly targeted treatment of patients infected with NTM.