Escherichia coli represents a significant concern for food safety, water quality, medical applications, and environmental health, necessitating effective detection methods. This study presents a reduced graphene oxide-based biosensor for the sensitive and specific detection of E. coli DNA. The functionalized reduced graphene oxide was successfully synthesized using a modified Hummers’ method, followed by a hydrothermal reduction step. Structural, morphological, and chemical properties of the reduced graphene oxide were confirmed through Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. The biosensor incorporates functionalized reduced graphene oxide linked to amino-modified probe DNA sequences specific to E. coli markers, providing stability and selective hybridization. Detection of E. coli DNA in the range of 0–476.19 fM is achieved by measuring absorbance changes at 273 nm, where an increase in absorbance indicates the presence of complementary E. coli single-stranded DNA. Performance testing with various DNA concentrations revealed a linear relationship, achieving a limit of detection of 80.28 fM and demonstrating high selectivity against non-target bacteria such as Bacillus subtilis, Enterococcus, Vibrio proteolyticus, and Staphylococcus. Optimization efforts identified the amino-E.coli BL21 probe as the only probe capable of detecting E. coli DNA. The reduced graphene oxide-based biosensor holds promise for rapid detection in pathogen monitoring, clinical diagnostics, and environmental analysis, paving the way for enhanced DNA biosensing technologies.
