Photon-upconversion nanoparticles for single-molecule biosensing and imaging

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FARKA Zdeněk MICKERT Matthias Jürgen GORRIS Hans-Heiner SKLÁDAL Petr

Year of publication 2021
Type Appeared in Conference without Proceedings
MU Faculty or unit

Faculty of Science

Description Fluorescence-based detection is a widespread readout method in immunoassays and cell imaging. However, it is limited by autofluorescence and light scattering. This optical background can be elegantly avoided by using photon-upconversion nanoparticles (UCNPs), which emit shorter-wavelength light under near-infrared excitation (anti-Stokes emission). These unique properties enabled us to image single UCNPs by wide-field microscopy and allowed the development of single-molecule (digital) immunoassays. We have synthesized conjugates of polyethylene glycol-coated UCNPs with streptavidin and applied them as a label to detect cancer biomarker prostate-specific antigen (PSA). The digital detection based on the counting of individual sandwich immunocomplexes provided a limit of detection (LOD) of 23 fg·ml-1 (800 aM) in 25% human serum, which is 20× more sensitive than the analog readout based on the measurement of output light intensity. The conjugates of UCNPs with streptavidin are also useful as a label in the diagnosis of bacterial diseases. We have developed immunoassays for the detection of bacteria Melissococcus plutonius and Paenibacillus larvae, the causative agents of European and American foulbrood, respectively. The assays provided LODs of 340 CFU·ml-1 and 2900 CFU·ml-1, and their practical applicability was verified by the analysis of real samples of bees, larvae, and bottom hive debris. Furthermore, the unique optical properties make UCNPs suitable for cell imaging. We have employed the PEG-based bioconjugates for the labeling of HER2 biomarker on breast cancer cells. The minimum optical background and low non-specific binding provided a superior signal-to-background ratio of 319, which is 50-fold better than conventional fluorescent labeling with the identical immunoreagents.
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