Publication details

Digital detection of subfemtomolar concentrations of prostate-specific antigen by single molecule immunosensing

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GORRIS Hans-Heiner MICKERT Matthias Jürgen FARKA Zdeněk KOSTIV Uliana HLAVÁČEK Antonín HORÁK Daniel SKLÁDAL Petr

Year of publication 2021
Type Conference abstract
MU Faculty or unit

Faculty of Science

Description The ability to detect single molecules holds the promise to reach the ultimate sensitivity in analytical chemistry. Several problems, however, must be addressed before single-molecule techniques can be employed for routine analytical applications. Photon-upconversion nanoparticles (UCNPs) are excellent labels for single molecule immunoassays because they emit short-wavelength light under near-infrared (NIR, 980 nm) excitation (anti-Stokes emission), which avoids autofluorescence and light scattering. These unique photoluminescent features enable the detection of UCNPs at the single nanoparticle level by conventional wide-field epiluminescence microscopy. Furthermore, very homogeneous UCNP labels were prepared by gel electrophoretic separation. For the upconversion-linked immunoassay (ULISA), microtiter plates were first coated with anti-prostate-specific antigen (PSA) antibodies to capture PSA. PSA was then detected either by an anti-PSA antibody-UCNP conjugate or a biotinylated anti-PSA antibody in combination with a streptavidin-PEG UCNP conjugate. The PSA concentration was determined on the same microtiter plate either (1) by using an upconversion microtiter plate reader (analog mode) or (2) by counting individual immunocomplexes under an upconversion wide-field microscope (digital mode). The lowest limit of detection was achieved by using the streptavidin-PEG-neridronate conjugate as a detection label. Counting single analyte molecules improved the limit of detection (LOD) (23 fg mL-1, 800 aM) by more than one order of magnitude compared to the analogue readout.
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