Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).

Department of Biochemistry Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Laboratory Methods Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Czech Republic

Institute of Analytical Chemistry Chemo and Biosensors University of Regensburg Universitätsstraße 31 930 53 Regensburg Germany

Institute of Analytical Chemistry of the Czech Academy of Sciences Veveří 97 602 00 Brno Czech Republic

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Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen