Amperometric Immunosensor for Rapid Detection of Honeybee Pathogen Melissococcus Plutonius

European foulbrood (EFB) is a honeybee larvae disease caused by a bacterium Melissococcus plutonius. An amperometric immunosensor based on a sandwich assay was developed for rapid point-of-care detection of this pathogen. An in-house made anti-Melissococcus antibody was immobilized to a gold surface of a screen-printed sensor via self-assembled monolayer of cysteamine activated with glutaraldehyde. The direct impedimetric detection of captured microbial cells was tested, however, a better performance was obtained after the formation of sandwich with the peroxidase-labeled antibody in the amperometric mode. The label-free assay was limited by higher non-specific binding. The limit of detection of the immunosensor was 6.6x10(4) CFU mL(-1) (colony-forming units) with wide linear range between 10(5) CFU mL(-1) and 10(9) CFU mL(-1). The whole analysis was completed within 2 h, which is shorter compared to common laboratory diagnostic tools, such as enzyme-linked immunosorbent assay or polymerase chain reaction. Furthermore, atomic force microscopy was used for confirmation of the bacteria presence on the electrode surface. The developed immunosensor was successfully employed in the analysis of real samples of honeybees and larvae. The achieved results demonstrate the potential of the amperometric immunosensor for practical in-field diagnosis of EFB, which can prevent infection spreading and connected losses of honeybee colonies.