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DNA damage induced by occupational exposure to copper oxide nanoparticles
Rössner st., Pavel ; Pelcová, D. ; Elzeinová, Fatima ; Mikuška, Pavel ; Večeřa, Zbyněk ; Coufalík, Pavel ; Vlčková, Š. ; Fenclová, Z. ; Rössnerová, Andrea
Copper oxide nanoparticles (CuO NPs) have a widespread use in industry, chemistry, in production of electronic devices and as an antimicrobial agent. Although copper is an important biogenic element, CuO NPs are toxic with the ability to induce oxidative stress, apoptosis, cell cycle arrest or DNA damage. For humans, the inhalation route is the most common way of exposure to CuO NPs. In the body, CuO NPs may be either deposited in the lungs, or transported to other organs. Their presence usually causes oxidative stress or inflammatory responses, consequently leading to DNA damage. In this study, we investigated the effect of CuO NPs inhalation on DNA damage in a group of researches conducting animal exposure experiments. The subjects were exposed to various metal oxide nanoparticles, including CuO NPs, by inhalation for an average of 4.9 ± 0.4 years. The average mass concentration of Cu in the air during the experiment was 7.3 ± 3.2 ng/m3. Subjects not exposed to nanoparticles served as a control group. We applied micronucleus assay using Human Pan Centromeric probes to detect DNA damage and to distinguish between the frequency of centromere positive (CEN+) and centromere negative (CEN−) micronuclei (MN) in the binucleated cells. We\ndid not find differences between both groups for either mean MN frequency (10.38 ± 2.50 vs. 11.88 ± 3.01 MN/1000 binucleated cells), or CEN+/CEN- ratio (58%/42% vs. 55%/45%), for the exposed and controls, respectively. In conclusion, inhalation of CuO NPs at this low-level exposure had no effect on chromosomal losses and/or breaks.
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Fast and highly sensitive laser scanner for recording photon-upconversion luminiscence from planar surfaces
Hlaváček, Antonín ; Křivánková, Jana ; Foret, František
Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanocrystals that can be excited by nearinfrared light and emit photon-upconversion luminescence of shorter wavelengths. Advantages of UCNPs include near-infrared excitation, multiple and narrow emission bands, negligible autofluorescence and high stability, which make UCNPs ideal luminescence label for use in biological and chemical assays. These assays - e.g. upconversion-linked immunosorbent assay, western blot, lateral flow assay, gel electrophoresis, thin layer chromatography - commonly require the scanning of a planar surface with a high spatial resolution and an excellent sensitivity. The availability of commercial equipment is recently limited because of the novelty of the photon-upconversion phenomenon. Therefore, we report on the construction of photon-upconversion laser scanner. The scanner consists of a laser scanning head, which is attached to a xy-moving stage. The scanning head itself is constructed as an epiluminescence detector with excitation wavelength of 976 nm. A CCD array spectroscope is connected to the laser head and serves as a sensitive detector of photon-upconversion luminescence. The scanner possesses a spatial resolution of 200 μm, the scanning rate is up to 57 points per second and the sensitivity reaches down to single photon-upconversion nanoparticle.
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Photon-upconversion nanoparticles for single-molecule immunosensing of cancer biomarkers and bacteria
Farka, Z. ; Mickert, M. J. ; Hlaváček, Antonín ; Poláchová, V. ; Kostiv, Uliana ; Pastucha, M. ; Horák, Daniel ; Gorris, H. H. ; Skládal, P.
The recent progress in the field of immunoassays has been driven by introduction of various kinds of nanomaterials. In particular, photon-upconversion nanoparticles (UCNPs) proved to be excellent immunoassay labels due to their ability to emit light of shorter wavelengths under near-infrared excitation (anti-Stokes emission), which prevents autofluorescence, minimizes light scattering, and thus reduces the optical background interference. These unique photoluminescent properties allow counting of individual biomolecules labeled with UCNPs by conventional wide-field epiluminescence microscopy and enable the development of single-molecule (digital) immunoassays. We have introduced a novel label based on UCNPs conjugated with streptavidin via poly(ethylene glycol) and applied it in a digital upconversion-linked immunosorbent assay (ULISA) for the detection of a cancer biomarker prostate specific antigen (PSA). The digital readout based on counting of individual immunocomplexes improved the sensitivity 16× compared to conventional analog readout and allowed to reach a limit of detection (LOD) of 23 fg·mL−1 (800 aM). Human serum samples were successfully analyzed achieving an excellent correlation with electrochemiluminescence reference method. The conjugates of UCNPs with streptavidin are also suitable for the detection of pathogenic bacterium Melissococcus plutonius, the causative agent of honeybee disease European foulbrood. The ULISA assay provided an LOD of 340 CFU·mL−1 and successfully analyzed real samples of bees, larvae and bottom hive debris. Due to the high reliability and relatively simple detection scheme, the digital ULISA can pave the way for a new generation of digital immunoassays with a strong potential for commercialization.
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Surface modification of nanoparticles for sustaining sensitivity of surface-enhanced raman spectrometric measurements in salinated environment
Týčová, Anna ; Přikryl, Jan ; Vaňhara, P. ; Klepárník, Karel ; Foret, František
Surface-enhanced Raman spectrometry (SERS) represents a powerful method for analysis of a broad spectrum of analytes ranging from inorganic ions to biomolecules of high complexity. It combines the potential\nof Raman spectrometry for a definite identification of an analyte with remarkable sensitivity achieved by the surface enhancement effect occurring on metal nanoparticles. While low ionic strength influences positively\nthe sensitivity of the SERS measurement, a higher level of inorganic salts leads to fast ruining of colloidal character, which completely devastates the effect of the surface enhancement. The common stabilization of\nnanoparticles by a layer of polymers has a negative impact on the SERS sensitivity since it shields the nanoparticle surface from the analytes. In this work, we aim at the development of the bi-ligand system of\nnanoparticles surface modification for improved stability of colloid in saline solution at sustaining the potential for sensitive SERS analyses. The proposed system relies on the binding of 3-mercaptopropionic acid and\nthiolated polyethylene glycol in a suitable ratio onto the nanoparticle surfaces. While the short chains of the acid sustain the accessibility of the surface for analytes, the polymeric structures act as a steric barrier\npreventing colloid aggregation.
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