Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers
| Authors | |
|---|---|
| Year of publication | 2017 |
| Type | Article in Periodical |
| Magazine / Source | Analytical Chemistry |
| MU Faculty or unit | |
| Citation | |
| web | http://pubs.acs.org/doi/10.1021/acs.analchem.7b03542 |
| Doi | http://dx.doi.org/10.1021/acs.analchem.7b03542 |
| Field | Analytic chemistry |
| Keywords | single-molecule analysis; upconversion-linked immunosorbent assay; cancer biomarker; prostate specific antigen; photon-upconversion nanoparticle |
| Description | 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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