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.

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

Institute of Analytical Chemistry Chemo and Biosensors University of Regensburg 93053 Regensburg Germany

Institute of Analytical Chemistry Czech Academy of Sciences 602 00 Brno Czech Republic

Institute of Macromolecular Chemistry Czech Academy of Sciences 162 06 Prague Czech Republic

References provided by Crossref.org

The pros and cons of nucleic acid-amplified immunoassays-a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification

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

Monodisperse Core-Shell NaYF4:Yb3+/Er3+@NaYF4:Nd3+-PEG-GGGRGDSGGGY-NH2 Nanoparticles Excitable at 808 and 980 nm: Design, Surface Engineering, and Application in Life Sciences