Electrocatalytic aptasensor for bacterial detection exploiting ferricyanide reduction by methylene blue on mixed PEG/aptamer monolayers

Pathogen-triggered infections are the most severe global threat to human health, and to provide their timely treatment and prevention, robust methods for rapid and reliable identification of pathogenic microorganisms are required. Here, we have developed a fast and inexpensive electrocatalytic aptamer assay enabling specific and ultrasensitive detection of E. coli. E. coli, a biomarker of environmental contamination and infections, was captured on the mixed aptamer/thiolated PEG self-assembled monolayers formed on electrochemically pretreated gold screen-printed electrodes (SPE). Signals from aptamer - E. coli binding were amplified by electrocatalytic reduction of ferricyanide mediated by methylene blue (MB) adsorbed on bacterial and aptamer surfaces. PEG operated as an antifouling agent and inhibited direct (not MB-mediated) discharge of ferricyanide. The assay allowed from 10 to 1000 CFU mL-1 E. coli detection in 30 min, with no interference from B. subtilis, in buffer and artificial urine samples. This electrocatalytic approach is fast, specific, sensitive, and can be used directly in infield and point-of-care applications for analysis of bacteria in human environment.