Amperometric Immunosensor for Rapid Detection of Honeybee Pathogen Melissococcus Plutonius

Abstract

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 6.6×104 CFU mL−1 (colony‐forming units) with wide linear range between 105 CFU mL−1 and 109 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.

Melissococcus plutonius Can Be Effectively and Economically Detected Using Hive Debris and Conventional PCR

“…Polachova et al [ 23 ] recently developed a highly sensitive up-conversion-linked immunosorbent assay (ULISA) that employs photon-up-conversion nanoparticles (UCNPs) for the detection of M. plutonius using polyclonal antibodies made in house. Biosensors have also recently been established in laboratory diagnostics [ 24 , 25 ]. The routine use of immune-based techniques is limited, however, because they require EFB specific antibodies.…”

Use of Cysteamine and Glutaraldehyde Chemicals for Robust Functionalization of Substrates with Protein Biomarkers—An Overview on the Construction of Biosensors with Different Transductions

“…Moreover, the LODs achieved in the complex matrices of homogenized bees and larvae were 2.4 × 10 5 and 7.0 × 10 5 CFU mL −1 , respectively. It is reported that negative control of P. alvei confirmed the high selectivity of such immunoassay [ 36 ]. Another study reported on the use of polyimide sheet modified with carbon and AuNPs inks through flexographic printing technique and drop casted with glucoseoxidase (5 μL, Gox, 7 × 10 9 pg/mL) for chronoamperometry detection of 26 μM glucose mutarotated in DI water for 24 h [ 37 ].…”

Immunosensors—The Future of Pathogen Real-Time Detection

“…This method is based on the measurement of a current flow that relates to the concentration of a measured analyte such as a pathogen. The amperometric platform applies a constant potential at the working electrode related to the reference electrode, where the potential is obtained from the electrochemical oxidation or electroreduction of an electroactive species [48,49]. This system has several advantages, including low costs and sensitivity.…”