gFET-based aptasensors technology allows sensitive and specific quantification of the ESKAPE pathogens

The rapid rise of antimicrobial resistance among the ESKAPE pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp., requires diagnostic technologies capable of fast, simplified and sensitive microbial detection. Conventional culture-based diagnostics remain too time-consuming to guide early therapeutic interventions. Here, we present a graphene field-effect transistor (gFET) aptasensor platform enabling rapid, label-free and highly sensitive quantification of all six ESKAPE pathogens. Each gFET device was functionalized with a specific DNA aptamer selected from literature sources and immobilized via a mixed pyrene-based linker strategy on reduced graphene oxide (rGO). Exposure of the functionalized sensors to logarithmically diluted bacterial suspensions (10-105 CFU mL-) produced characteristic and concentration-dependent shifts in source-drain current ({Delta}IDS). For all pathogen-specific aptamers, {Delta}IDS correlated linearly with bacterial load (R2 = 0.90-1.00), while non-target bacteria generated only low-level, unspecific fluctuations. Limits of detection ranged from 10 to 1000 bacterial cells depending on the aptamer. Together, these results demonstrate that aptamer-functionalized rGO-FETs provide a robust, scalable and highly specific electronic biosensing architecture capable of distinguishing clinically relevant multidrug-resistant pathogens with excellent analytical performance. Author summaryAntimicrobial-resistant bacteria pose a growing threat to global health, especially the so-called ESKAPE pathogens, which frequently cause hospital-acquired infections and are increasingly difficult to treat. Current diagnostic methods can take several days, delaying the start of effective therapy. In our work, we developed a fast and highly sensitive biosensor that uses electrically conductive GO and short DNA molecules, called aptamers, to recognize specific bacteria. When a pathogen binds to its matching aptamer on the sensor surface, the electrical signal of the graphene changes in a measurable way. We tested ESKAPE species and showed that all of the investigated aptamers detect only their intended bacterial targets, even at very low concentrations. Importantly, the sensors respond within minutes and do not require any labelling or complex sample preparation. Our technology demonstrates how graphene-based aptasensors can support rapid and accurate detection of dangerous bacterial pathogens and could ultimately help clinicians make faster decisions in treating infections.