Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay

Abstract

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.

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

“…Oleic acid (OA), hexane, methanol, ethanol, and acetone were purchased from Lach-Ner (Neratovice, Czech Republic). Sodium neridronate and neridronate-PEG-alkyne (Ner-PEG-Alk) were prepared as described earlier (Kostiv et al, 2017c;Mickert et al, 2019). Azidopentanoyl-GGGRGDSGGGY-NH 2 (RGDS) peptide was synthesized by the standard Fmoc/tBu solid-phase technique on a TentaGel R Rink-Amide-R resin according to previously published procedures (Proks et al, 2012;Kostiv et al, 2016).…”

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

“…The number of UCNPs is given by the diffraction-limited spots in (B) and the upconversion luminescence (UCL) is recorded by amicrotiter plate reader. [22] than two NPs.T osuppress these effects,large intensity bursts that were attributed to aggregates were removed before calculating the auto-and cross-correlation functions. [103] However,t he smaller bursts were still causing strong variations of the amplitude,w hich affected the assay sensitivity.…”

“…[21] Theb ackground-free detection renders UCNPs an excellent candidate for single-molecule applications. [22] Furthermore,n anocontainers such as liposomes can be filled with large numbers of fluorophores to generate as trong signal. In contrast to enzyme labels that generate the fluorophores in situ, the encapsulated fluorophores are released on demand from the nanocontainer to avoid self-quenching inside the confined environment.…”

The Growing Impact of Micro/Nanomaterial‐Based Systems in Precision Oncology: Translating “Multiomics” Technologies

“…Reproduced with permission. [66] Copyright 2019, American Chemical Society. c) Gold core-silver shell pearl nanoparticles as Raman signal enhancing substrates for highly sensitive detection of pancreatic cancer-derived exosomes.…”