| Identification of pathogens and diagnosis of infections are imperative health challenges, mainly in the case of fastidious bacteria that are hard to grow. DNA-based biosensors, including gene chips, are of major interest due to their tremendous promise for detecting diseases and obtaining sequence-specific information in a faster, simpler and cost-effective manner compared to the traditional hybridization techniques. In this study, an innovative conductive nano-ink based on graphite/silver nanoparticles (AgNPs) was synthesized and applied towards electroanalytical biosensing of bacteria genome. The prepared nano-ink was drop-casted on the glassy-surface carbon electrode and used for the bioassay of L. pneumophila in real samples obtained from humans. Furthermore, toluidine blue was used as a redox indicator for monitoring the nucleic acid hybridization. So, a conductive ink-based biosensor for the electroanalytical biosensing of L. pneumophila mip gene was constructed by a simple and cost-benefit method. Chronoamperometry technique was applied for the electroanalytical investigation at optimum conditions. The obtained results showed that the proposed nano-ink had excellent conductivity towards electrode modification and sensor construction. Therefore, the proposed interface was applied to DNA hybridization in which toluidine blue was used as a redox hybridization indicator. In addition, the fabricated biosensor showed excellent sensitivity and good selectivity to the one-base and three-base mismatched ssDNA. The acquired results verified that in the ideal conditions, the linear range was 1 μM to 1 ZM and the low limit of quantification was 1 ZM. Finally, graphite–carbon based geno-ink was produced and used for the sensing of target DNA. Consequently, it is apparent that carbon–silver nanoparticles as a novel conductive nano-ink is an ideal material for electrode modification. |