Informace o publikaci

Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay


MICKERT Matthias Jürgen FARKA Zdeněk KOSTIV Uliana HLAVÁČEK Antonín HORÁK Daniel SKLÁDAL Petr GORRIS Hans-Heiner

Druh Článek v odborném periodiku
Časopis / Zdroj Analytical Chemistry
Fakulta / Pracoviště MU

Středoevropský technologický institut

Klíčová slova photon-upconversion nanoparticle; bioconjugation; single-molecule immunoassay; digital detection; prostate-specific antigen; cancer biomarker
Popis Single-molecule (digital) immunoassays provide the ability to detect much lower protein concentrations than conventional immunoassays. As photon-upconversion nanoparticles (UCNPs) can be detected without optical background interference, they are excellent labels for so-called single-molecule upconversion-linked immunosorbent assays (ULISAs). We have introduced a UCNP label design based on streptavidin-PEG-neridronate and a two-step detection scheme involving a biotinylated antibody that efficiently reduces nonspecific binding on microtiter plates. In a microtiter plate immunoassay, individual sandwich immune complexes of the cancer marker prostate-specific antigen (PSA) are detected and counted by wide-field epiluminescence microscopy (digital readout). The digital detection is 16× more sensitive than the respective analogue readout and thus expands the limit of detection to the sub-femtomolar concentration range (LOD: 23 fg mL–1, 800 aM). The single molecule ULISA shows excellent correlation with an electrochemiluminescence reference method. Although the analogue readout can routinely measure PSA concentrations in human serum samples, very low concentrations have to be monitored after radical prostatectomy. Combining the digital and analogue readout covers a dynamic range of more than 3 orders of magnitude in a single experiment.
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