Publication details

Photon-upconversion nanoparticles as a background-free label for the detection of biomarkers and bacteria

Authors

ODSTRČILÍKOVÁ Eliška PASTUCHA Matěj HLAVÁČEK Antonín SKLÁDAL Petr FARKA Zdeněk

Year of publication 2020
Type Article in Proceedings
Conference XX. Workshop of Biophysical Chemists and Electrochemists
MU Faculty or unit

Faculty of Science

Citation
Web https://www.sci.muni.cz/labifel/files/soubory/sbornik_2020.pdf#page=37
Description Due to their high specificity, immunochemical assays are widely used for the detection of various analytes within complex matrices. The most commonly used immunoanalytical method – enzyme-linked immunosorbent assay (ELISA) – relies on signal generation by enzymes, typically horseradish peroxidase. The enzymes provide highly sensitive detection because a single enzyme molecule can transform numerous molecules of the substrate. However, enzymes also suffer several disadvantages, most notably low stability. Recent progress in nanotechnology has provided various nanomaterials, which can be used as labels in immunoassays. In particular, photon-upconversion nanoparticles (UCNP) are useful as alternative detection labels because their anti-Stokes luminescence can be excited by the NIR laser and detected in the Vis region without optical background interference. However, the as-synthesized UCNPs are unstable in high ionic strength buffers, lack suitable functional groups, and are prone to nonspecific interactions in complex samples. Hence, their surface needs to be modified for successful biological applications. The most common methods include silanization or modification by ligands that can coordinate to the lanthanide ions on the UCNP surface. We have introduced a novel approach for the conjugation of biomolecules with UCNPs based on heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide. The alkyne groups were used to conjugate the UCNPs with streptavidin and anti-human serum albumin antibody via copper-catalyzed click chemistry. The alternative modification approach consisted of a mild reduction of the disulfide bonds of the antibody and conjugation via maleimide. The nanoconjugates were used as labels for an upconversion-linked immunosorbent assay (ULISA) for the detection of human serum albumin (HSA), which is a marker of kidney disfunction. The streptavidin-based labels achieved the LOD of 0.17 ng/mL for the target HSA, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL). The optimized assay was also employed for the analysis of real samples of spiked urine, demonstrating the practical potential of the method. The conjugates of UCNPs are also suitable for the detection of bacteria. We have developed an ULISA assay for Melissococcus plutonius, the causative agent of honeybee disease European foulbrood. The assay provided an LOD of 340 CFU/mL and was successfully employed in the analysis of real samples of bees, larvae, and bottom hive debris. Due to the high reliability and relatively simple detection scheme, the ULISA assays can pave the way for a new generation of immunoassays with a strong potential for commercialization.
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