A method for the conjugation of photon-upconversion nanoparticles with streptavidin via copper-free click-chemistry was introduced, and the prepared label was applied in an immunoassay for European foulbrood diagnosis.
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.
The recent progress in the field of immunoassays has been driven by introduction of various kinds of nanomaterials. In particular, photon-upconversion nanoparticles (UCNPs) proved to be excellent immunoassay labels due to their ability to emit light of shorter wavelengths under near-infrared excitation (anti-Stokes emission), which prevents autofluorescence, minimizes light scattering, and thus reduces the optical background interference. These unique photoluminescent properties allow counting of individual biomolecules labeled with UCNPs by conventional wide-field epiluminescence microscopy and enable the development of single-molecule (digital) immunoassays. We have introduced a novel label based on UCNPs conjugated with streptavidin via poly(ethylene glycol) and applied it in a digital upconversion-linked immunosorbent assay (ULISA) for the detection of a cancer biomarker prostate specific antigen (PSA). The digital readout based on counting of individual immunocomplexes improved the sensitivity 16× compared to conventional analog readout and allowed to reach a limit of detection (LOD) of 23 fg•mL −1 (800 aM). Human serum samples were successfully analyzed achieving an excellent correlation with electrochemiluminescence reference method. The conjugates of UCNPs with streptavidin are also suitable for the detection of pathogenic bacterium Melissococcus plutonius, the causative agent of honeybee disease European foulbrood. The ULISA assay provided an LOD of 340 CFU•mL −1 and successfully analyzed real samples of bees, larvae and bottom hive debris. Due to the high reliability and relatively simple detection scheme, the digital ULISA can pave the way for a new generation of digital immunoassays with a strong potential for commercialization.
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