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Single-molecule immunoassays based on upconversion nanoparticles for detection of cancer markers and bacteria

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FARKA Zdeněk MICKERT Matthias Jürgen HLAVÁČEK Antonín KOSTIV Uliana POLÁCHOVÁ Veronika PASTUCHA Matěj GORRIS Hans-Heiner SKLÁDAL Petr

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Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
Popis Single-molecule immunoassays have recently attracted wide attention due to the ability to identify and detect individual analyte molecules, which provides the ultimate sensitivity in bioanalytical applications. Popular labels for single-molecule assays are enzymes in confined volumes, however, the signal generation is time consuming, enzymes have limited stability and the supporting chips are often costly. In recent years, many techniques based on nanomaterials as detection labels have emerged, that can be used to overcome the disadvantages of enzymes and improve the assay properties. Photon-upconversion nanoparticles (UCNPs) are excellent labels for single-molecule immunoassays because they emit shorter-wavelength light under near-infrared excitation (anti-Stokes emission), which limits autofluorescence and light scattering. These unique photoluminescent features enable the detection of UCNPs at the single nanoparticle level by conventional wide-field epiluminescence microscopy. We have synthesized streptavidin-conjugated polyethylene glycol-coated UCNPs and applied them as labels for the detection of the cancer biomarker prostate specific antigen (PSA). The individual sandwich immunocomplexes were counted using a wide-field microscope equipped with a 980 nm laser excitation source. The noise-surpassing digital readout (particle counting) resulted in 20× higher sensitivity than the analog readout (emitted light intensity) and allowed to reach a detection limit of 800 aM (23 fg mL-1) in 25% serum. The streptavidin-conjugated UCNPs are also suitable as labels for detection of pathogenic bacteria. We have developed an assay based on in-house prepared antibodies for the detection of Melissococcus plutonius, causative agent of European foulbrood. The assay provided detection limit of 10^4 CFU mL-1 and its practical relevance was demonstrated by the analysis of real samples of honeybees and larvae.
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