Single molecule upconversion-linked immunosorbent assay with extended dynamic range for the sensitive detection of diagnostic biomarkers

0481051 - ÚIACH 2018 RIV US eng J - Journal Article
Farka, Z. - Matthias, J. M. - Hlaváček, Antonín - Skládal, P. - Gorris, H H.
Single molecule upconversion-linked immunosorbent assay with extended dynamic range for the sensitive detection of diagnostic biomarkers.
Analytical Chemistry. Roč. 89, NOV (2017), s. 11825-11830. ISSN 0003-2700. E-ISSN 1520-6882
R&D Projects: GA ČR(CZ) GBP206/12/G014
Institutional support: RVO:68081715
Keywords : photon upconversion * immunoassay * single molecule detection
OECD category: Analytical chemistry
Impact factor: 6.042, year: 2017

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.
Permanent Link: http://hdl.handle.net/11104/0276665