Publication details

Single-molecule upconversion-linked digital immunosensing of cancer biomarkers

Authors

FARKA Zdeněk MICKERT Matthias J. HLAVÁČEK Antonín GORRIS Hans H. SKLÁDAL Petr

Year of publication 2018
Type Conference abstract
MU Faculty or unit

Central European Institute of Technology

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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