Publication details

Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers

Authors

FARKA Zdeněk MICKERT Matthias J. HLAVÁČEK Antonín SKLÁDAL Petr GORRIS Hans H.

Year of publication 2017
Type Article in Periodical
Magazine / Source Analytical Chemistry
MU Faculty or unit

Central European Institute of Technology

Citation
Web http://pubs.acs.org/doi/10.1021/acs.analchem.7b03542
Doi http://dx.doi.org/10.1021/acs.analchem.7b03542
Field Analytic chemistry
Keywords single-molecule analysis; upconversion-linked immunosorbent assay; cancer biomarker; prostate specific antigen; photon-upconversion nanoparticle
Description The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL–1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.
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