Click-conjugated photon-upconversion nanoparticles in an immunoassay for honeybee pathogen Melissococcus plutonius

European foulbrood (EFB) is an infectious disease affecting honeybee larvae caused by the bacterium Melissococcus plutonius. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for antibody-based bacteria detection, however, its sensitivity is not high enough to reveal early-stage EFB infection. Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanomaterials that emit light of shorter wavelength under near-infrared (NIR) excitation and thus avoid optical background interference. After conjugation with specific biorecognition molecules, UCNPs can be used as ultrasensitive labels in immunoassays. Here, we introduce a method for conjugation of UCNPs with streptavidin based on copper-free click chemistry, which involves surface modification of UCNPs with alkyne-modified bovine serum albumin (BSA) that prevents the non-specific binding and provides reactive groups for conjugation with streptavidin-azide. To develop a sandwich upconversion-linked immunosorbent assay (ULISA) for M. plutonius detection, we have prepared a rabbit polyclonal anti-Melissococcus antibody. The specific capture of the bacteria was followed by binding of biotinylated antibody and UCNP-BSA-streptavidin conjugate for a highly sensitive upconversion readout. The assay yielded an LOD of 340 CFU mL-1 with a wide working range up to 109 CFU mL-1, which is 400 times better than the LOD of the conventional ELISA. The practical applicability of the ULISA was successfully demonstrated by detecting M. plutonius in spiked real samples of bees, larvae and bottom hive debris. These results show a great potential of the assay for early diagnosis of EFB, which can prevent uncontrolled spreading of the infection and losses of honeybee colonies.