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Nanoparticle-based immunochemical assays: new twist to classic analytical tools

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

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Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
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 improve the assay properties. The applications of nanoparticle labels in immunoassays can be divided into two categories: (a) catalytic nanoparticles (nanozymes) that provide transformation of substrate and (b) labels that can be detected directly, such as luminescent or metal nanoparticles. In this contribution, new nano-analytical techniques taking full advantage of catalytic and luminescent nanoparticles will be described. We have pioneered the use of Prussian blue nanoparticles (PBNPs) as label in nanozyme-linked immunosorbent assay (NLISA). The PBNPs are 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 a method for conjugation of PBNPs with antibodies and demonstrated the universal applicability of the label by the development of two sandwich immunoassays. 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 for direct detection on microtiter plate 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 a direct competitive upconversion-linked immunosorbent assay (ULISA) for the detection of pharmaceutical diclofenac, which is a common micropollutant in waters. The single-step assay had LOD of 20 pg mL-1 and the analysis time of only 70 min. The low background and high photostability make UCNPs a powerful tool for single molecule immunoassays. We have developed an optical approach for visualizing individual UCNPs by conventional 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.
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