Publication details
Single-molecule upconversion-linked digital immunosensing of cancer biomarkers
| Authors | |
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| Year of publication | 2018 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | The ability to detect cancer biomarkers at the single-molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interferences, and therefore is typically not suitable for real world analytical applications. This optical background can be elegantly avoided by using photon-upconversion nanoparticles (UCNPs) that emit shorter-wavelength light under near-infrared excitation (anti-Stokes emission). Conjugates of UCNPs with antibody can be used as a background-free luminescent tracer in upconversion-linked immunoassays. We have developed an optical approach for visualizing individual UCNPs and applied it for the sensitive detection of the cancer biomarker prostate specific antigen (PSA). An inverted wide-field microscope was equipped with a fiber-coupled 980 nm continuous-wave laser diode, 100× heat-resistant objective and a sensitive sCMOS camera. Silica-coated UCNPs with carboxyl groups on the surface were synthesized and conjugated with anti-PSA antibody. The structure and monodispersity of the nanoconjugates was studied in detail by transmission electron microscopy and agarose gel electrophoresis. Individual sandwich immunocomplexes consisting of (1) immobilized anti-PSA antibody, (2) PSA, and (3) anti-PSA antibody-UCNP conjugate were counted under an upconversion wide-field microscope equipped with a 980 nm laser excitation source. The noise-surpassing digital readout provided single particle resolution with almost no instrumental background. This allowed to reach a limit of detection (LOD) of 1.2 pg mL-1 (42 fM) of PSA in 25 % blood serum which is about ten times more sensitive than a classical (analog) readout of luminescence intensity. The digital signal readout is a completely new approach with the potential to pave the way for a new generation of immunoassays. |
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