The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

• MeSH
• COVID-19 * diagnóza   MeSH
• ELISA MeSH
• imunosorbenty * MeSH
• lidé MeSH
• nukleokapsida - proteiny MeSH
• protilátky virové MeSH
• SARS-CoV-2 MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunosorbenty * MeSH
• nukleokapsida - proteiny MeSH
• protilátky virové MeSH

The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.

• MeSH
• imunosorbenty MeSH
• lidé MeSH
• nádorové biomarkery MeSH
• nádory * diagnóza   MeSH
• nanočástice * chemie   MeSH
• oxid křemičitý chemie   MeSH
• polyethylenglykoly chemie   MeSH
• streptavidin MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• imunosorbenty MeSH
• nádorové biomarkery MeSH
• oxid křemičitý MeSH
• polyethylenglykoly MeSH
• streptavidin MeSH

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL-1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals.

• Klíčová slova
• anti-Stokes emission, cardiac arrest, lanthanide-doped nanomaterials, single molecule immunoassay, troponin,
• MeSH
• fotony MeSH
• imunoanalýza MeSH
• lidé MeSH
• nanočástice * MeSH
• troponin MeSH
• velikost částic MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• troponin MeSH