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

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 re...

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Published inAnalytical chemistry (Washington) Vol. 89; no. 21; pp. 11825 - 11830
Main Authors Farka, Zdeněk, Mickert, Matthias J, Hlaváček, Antonín, Skládal, Petr, Gorris, Hans H
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 07.11.2017
Subjects
Biomarkers
Biomarkers - analysis
Cancer
Chemistry
Counting
Diagnostic systems
Dynamic range
Fluorescence
Immunoassay
Immunoassay - methods
Immunoassays
Immunosorbents - chemistry
Immunosorbents - immunology
Limit of Detection
Luminescence
Molecules
Nanoparticles
Nanoparticles - chemistry
Prostate cancer
Prostate-Specific Antigen - analysis
Sensitivity analysis
Upconversion
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Summary: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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ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.7b03542