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Nanoparticle-based immunochemical assays: Nano-update of classic analytical tools


FARKA Zdeněk HLAVÁČEK Antonín MICKERT Matthias Jürgen POLÁCHOVÁ Veronika GORRIS Hans-Heiner SKLÁDAL Petr

Druh Článek ve sborníku
Konference XVIII. Workshop of Biophysical Chemists and Electrochemists
Fakulta / Pracoviště MU

Středoevropský technologický institut

Klíčová slova Immunoassay; Single-molecule analysis; Photon-upconversion nanoparticle; Prussian blue nanoparticle; Bioconjugation
Popis Enzyme immunoassays are widely used for detection of various analytes within complex samples. However, enzymes as labels suffer several disadvantages such as high production costs, limited stability, and time-consuming signal development. The recent progress in nanotechnology has led to the development of various nanomaterials that can be used to overcome the disadvantages of enzymes and improve the assay properties. Prussian blue nanoparticles (PBNPs) are nanozymes composed of the Fe4[Fe(CN)6]3-based coordination polymer. They reveal peroxidase-like activity and are capable of catalyzing the oxidation of colorless 3,3',5,5'-tetramethylbenzidine to form intensely blue product. We have introduced the method for conjugation of PBNPs with antibodies and demonstrated the universal applicability of the label by the development of two sandwich nanozyme-linked immunosorbent assays (NLISAs). Human serum albumin was detected in urine for the diagnosis of albuminuria with a limit of detection (LOD) of 1.2 ng mL-1 and bacterial pathogen Salmonella Typhimurirum was detected in powdered milk with LOD of 6·10^3 CFU mL-1. Photon-upconversion nanoparticles (UCNPs) are particularly useful as immunoassay labels because their anti-Stokes luminescence can be excited by the NIR laser and detected in the VIS region without optical background interference. We have developed indirect and direct competitive upconversion-linked immunosorbent assay (ULISA) for the detection of pharmaceutical diclofenac, which is a common micropollutant in waters. The direct single-step assay has LOD of 20 pg mL-1 and the analysis time of 70 min. The low background and high photostability make UCNPs a powerful tool for detection of single molecules. We have developed an optical approach for visualizing individual UCNPs by epiluminescence microscopy and applied it for the sensitive detection of the cancer biomarker prostate specific antigen (PSA). Individual sandwich immunocomplexes were counted under an upconversion 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 an LOD of 1.2 pg mL-1 (42 fM) of PSA in 25% blood serum which is about ten times more sensitive than commercial immunoassays. The concept of introducing nanomaterials into immunoassays modernizes the classic bioanalytical methods and improves their working parameters. 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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