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.

CEITEC Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

Institute of Analytical Chemistry Chemo and Biosensors University of Regensburg 93040 Regensburg Germany

Institute of Analytical Chemistry of the Czech Academy of Sciences v v i 602 00 Brno Czech Republic

Citace poskytuje Crossref.org

Amplification-Free Attomolar Detection of Short Nucleic Acids with Upconversion Luminescence: Eliminating Nonspecific Binding by Hybridization Complex Transfer

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design

Bright photon upconversion in LiYbF4:Tm3+@LiYF4 nanoparticles and their application for singlet oxygen generation and in immunoassay for SARS-CoV-2 nucleoprotein

Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay

Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging

Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

Laser-induced breakdown spectroscopy as a readout method for immunocytochemistry with upconversion nanoparticles

Advances in Optical Single-Molecule Detection: En Route to Supersensitive Bioaffinity Assays

Laser-induced breakdown spectroscopy as a novel readout method for nanoparticle-based immunoassays