amyloid, fibrillization, fluorescence, atomic force microscopy, nanoparticles
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Scopus Publications
Scopus Publications
A novel approach to the photochemical UV-C activation of PAA-coated Fe₃O₄ nanoparticles for oriented APTES functionalisation with free surface-amino groups M. Barutiak, A. Zeleňáková, P. Hrubovčák, L. Nagy, D. Volavka, J. Szűcsová, Ž. Fabriciová, A. Antošová Applied Surface Science Advances, 2026 • Fe₃O₄ nanoparticles with polyhedral and cubic morphologies were synthesised by thermal decomposition. • A novel method was used for surface modification of Fe 3 O 4 @PAA MNPs with APTES. • ATR-FTIR and XPS confirmed successful ligand attachment; zeta potential shifted from negative to positive. • RNA extraction efficiency of modified nanoparticles was comparable to SiO₂-coated MNPs commonly used in commercial-like kits. Fe₃O₄ MNPs with polyhedral ( P1 ) and cubic ( K1 ) morphologies, prepared by a thermal decomposition method, were coated with polyacrylic acid (PAA) and surface functionalised with the organic ligand 3-(aminopropyl)triethoxysilane (APTES). A novel method was used to attach APTES to polyacrylic acid, where we used photochemical activation of the PAA layer by UV-C (254 nm). The novelty of this work lies in the simple yet unique UV-C activation of surface oxyl groups to achieve oriented APTES functionalisation. The surface functionalisation, together with controlled nanoparticle morphology, was intended to improve the affinity of magnetic nanoparticles (MNPs) for nucleic acids and thus facilitate efficient nucleic acid separation. Structural and surface characterisation was performed using XRD, SEM, ATR-FTIR, XPS, and zeta potential analysis, confirming the successful modification of the surface of nanoparticles with APTES. SQUID magnetometry analysis demonstrated morphology-dependent magnetic response. Polyhedral Fe₃O₄ nanoparticles exhibited a higher saturation magnetisation (∼90 Am² kg⁻¹) compared to their cubic counterparts (∼70 Am² kg⁻¹), reflecting differences in magnetic anisotropy and structural ordering. Both morphological variants show the Verwey transition near 120 K, indicative of high crystallinity and phase purity of the magnetite core. Magnetic measurements using SQUID magnetometry revealed multidomain behaviour at room temperature, essential for magnetic separation applications. RNA extraction tests demonstrated that P1 and K1 nanoparticles exhibited separation efficiency that is comparable to spherical Fe₃O₄@SiO₂ particles used in commercial kits, despite lacking a silica layer. These findings highlight the potential of Fe₃O₄ MNPs with diverse morphology and ligand-functionalised nanoparticles as the basis for next-generation nucleic acid isolation systems for different advanced biomedical applications.
Enhanced efficiency of Fe3O4@SiO2 with specific ligand layer in magnetic separation technique M. Barutiak, A. Zeleňáková, P. Hrubovčák, L. Nagy, J. Szűcsová, et al. Surfaces and Interfaces, 2025 We have designed core-shell nanoparticles that enhance the separation efficiency of nucleic acids and genetic material during RT-PCR assays. The improvement has been achieved by using a specific ligand modification of the nanoparticles’ surface. Fe 3 O 4 @SiO 2 nanoparticles with a magnetic core size of around 10 nm and a silica shell were modified with an outer layer of organic ligand. In this way, four different samples were prepared, consisting of the same core-shell Fe 3 O 4 @SiO 2 system with different ligands attached to the surface. The specific ligands were chosen to enhance the RNA binding efficiency and differed from each other by functional groups. We characterised the prepared nanoparticle systems and studied their structural, morphological, and magnetic properties and their potential to bind nucleic acids by the extraction protocol and ethidium bromide fluorescence displacement assay. Our study shows that the modification of the nanoparticle surface with a suitable ligand results in a significant increase in the separation efficiency of RNA molecules.
Experimental Study of Magnetic Nanoparticles for Magnetic Separation DNA/RNA M. Barutiak, A. Zeleňáková, P. Hrubovčák, Ľ. Nagy, Ž Fabriciová, M. Lisnichuk, E. Beňová, N. Király, V. Zeleňák, A. Antošová, A. Jacková, Š. Vilček IEEE Transactions on Magnetics, 2025 This work deals with the magnetic characterization of magnetic nanoparticles (NPs) as nucleic acid separation reagents in RT-PCR assays. We have designed a core-multishell NP system that with the aid of a specific ligand on the NP surface increases the efficiency of the separation of nucleic acids and genetic material during RT-PCR testing. The Fe3O<inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$_{\mathbf {4}}$ </tex-math></inline-formula>-SiO2 core-shell NPs with magnetic core size 10 nm and silica shell thickness 3 nm have been modified by the outer layer consisting of 3-(trimethoxysilyl)propyl methacrylate (TPMC). The specific surface layer provides a larger area and higher number of binding centers for DNA/RNA when compared with commercial Fe3O4-SiO2 Kit. Our study shows that despite of significant increase in NP size after ligand functionalization (<inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$D \sim 22$ </tex-math></inline-formula> nm), the feasible magnetic properties have been preserved and are similar to commercial Kit. With the conservation of magnetic properties and improvement of binding potential, we have obtained enhanced efficiency in the separation of DNA/RNA molecules when compared to commercial NPs.
Anti-amyloid activity of amino acid functionalized magnetic nanoparticles on αLactalbumin aggregation Andrea Antosova, Miroslav Gancar, Zuzana Bednarikova, Iryna Antal, Patrizia Verducci, Olga Parmar, Martina Kubovcikova, Martina Koneracka, Vlasta Zavisova, Patrizio Graziosi, Barbara Luppi, Zuzana Gazova, Eva Bystrenova Nano Structures and Nano Objects, 2024 Protein amyloid aggregation involves structural changes in native protein conformers and the formation of amyloid fibrils that accumulate in deposits in the human body. This study explores the effect of magnetic nanoparticles functionalized with amino acids (aaMNPs)—cysteine (Cys), poly-L-lysine (PLL), or proline (Pro)—on the amyloid aggregation of α-lactalbumin (αLA) and its amyloid fibrils (LAF). Our results from thioflavin T fluorescence assay (ThT), atomic force microscopy (AFM), and infrared spectroscopy revealed that the studied aaMNPs inhibit αLA fibrillization and destruct LAF in a concentration-dependent manner. The type of amino acid used for nanoparticle functionalization significantly influences the anti-amyloid efficacy. ProMNPs exhibit the highest inhibitory activity, with the timing of their addition being crucial Conversely, CysMNPs demonstrate the highest destructing activity. AFM image analysis through grain mapping was employed to quantify the anti-amyloid effects of aaMNPs. Cytotoxicity testing on kidney cells identified PLLMNPs as the only cytotoxic nanoparticles in our study. These findings clarify the mechanisms of inhibition and destruction of LAF in the presence of aaMNPs, which could inform the design of nanoparticles for therapeutic purposes in the future.
Silica-magnetite nanoparticles: Synthesis, characterization and nucleic acid separation potential Zuzana Bednarikova, Martina Kubovcikova, Iryna Antal, Andrea Antosova, Miroslav Gancar, Jozef Kovac, Radka Sobotova, Vladimir Girman, Diana Fedunova, Martina Koneracka, Zuzana Gazova, Vlasta Zavisova Surfaces and Interfaces, 2023 Silica-coated magnetic nanoparticles can be utilized for the magnetic separation of nucleic acids, which is an important step in many clinical procedures. A series of silica-functionalized superparamagnetic iron oxide nanoparticles (SiO2@MNPs) was synthesized to efficiently separate nucleic acids for the sensitive detection of viral infections. Magnetite nanoparticles (MNPs) coated with different thicknesses of silica layer resulting from different ratios of tetraethylorthosilicate (TEOS) precursor to magnetite (TEOS/MNPs = 0.4, 1.0, 2.0, 3.0, 4.0, 5.0 ml/g) have been selected for characterization of their physico-chemical properties and biological activities. The SiO2@MNPs' morphology, size, magnetic and surface properties, cytotoxicity and nucleic acid binding propensity were determined to select the ones with the highest RNA separation potential. Our results showed that RNA-binding properties and magnetic separation efficiency are nanoparticle size-dependent. The highest binding and separation efficiencies were detected for the smaller SiO2@MNPs with TEOS/MNPs (v/w) ratios equal to 1.0 and 2.0. These nanoparticles are promising candidates for clinical application relevant to RNA viruses.
Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2 Veronika Džupponová, Nataša Tomášková, Andrea Antošová, Erik Sedlák, Gabriel Žoldák International Journal of Molecular Sciences, 2023 Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.
The influence of cations on α-lactalbumin amyloid aggregation Andrea Antosova, Miroslav Gancar, Zuzana Bednarikova, Jozef Marek, Eva Bystrenova, Zuzana Gazova Journal of Biological Inorganic Chemistry, 2022 There is limited knowledge regarding α-lactalbumin amyloid aggregation and its mechanism. We examined the formation of α-lactalbumin amyloid fibrils (α-LAF) in the presence of cations (Mg2+, Ca2+, Na+, K+, NH4+, and Cs+) in the form of chloride salts at two concentrations. We have shown that studied cations affect the conformation of α-lactalbumin, the kinetics of its amyloid formation, morphology, and secondary structure of α-LAF in a different manner. The higher salts concentration significantly accelerated the aggregation process. Both salt concentrations stabilized α-lactalbumin's secondary structure. However, the presence of divalent cations resulted in shorter fibrils with less β-sheet content. Moreover, strongly hydrated Mg2+ significantly altered α-lactalbumin's tertiary structure, followed by Na+, NH4+, K+, and weakly hydrated Cs+. On the other hand, Ca2+, despite being also strongly hydrated, stabilized the tertiary structure, supposedly due to its high affinity towards α-lactalbumin. Yet, Ca2+ was not able to inhibit α-lactalbumin amyloid aggregation. Graphic abstract
Effect of 1-Ethyl-3-methylimidazolium Tetrafluoroborate and Acetate Ionic Liquids on Stability and Amyloid Aggregation of Lysozyme Diana Fedunova, Andrea Antosova, Jozef Marek, Vladimir Vanik, Erna Demjen, Zuzana Bednarikova, Zuzana Gazova International Journal of Molecular Sciences, 2022 Amyloid fibrils draw attention as potential novel biomaterials due to their high stability, strength, elasticity or resistance against degradation. Therefore, the controlled and fast fibrillization process is of great interest, which raises the demand for effective tools capable of regulating amyloid fibrillization. Ionic liquids (ILs) were identified as effective modulators of amyloid aggregation. The present work is focused on the study of the effect of 1-ethyl-3-methyl imidazolium-based ILs with kosmotropic anion acetate (EMIM-ac) and chaotropic cation tetrafluoroborate (EMIM-BF4) on the kinetics of lysozyme amyloid aggregation and morphology of formed fibrils using fluorescence and CD spectroscopy, differential scanning calorimetry, AFM with statistical image analysis and docking calculations. We have found that both ILs decrease the thermal stability of lysozyme and significantly accelerate amyloid fibrillization in a dose-dependent manner at concentrations of 0.5%, 1% and 5% (v/v) in conditions and time-frames when no fibrils are formed in ILs-free solvent. The effect of EMIM-BF4 is more prominent than EMIM-ac due to the different specific interactions of the anionic part with the protein surface. Although both ILs induced formation of amyloid fibrils with typical needle-like morphology, a higher variability of fibril morphology consisting of a different number of intertwining protofilaments was identified for EMIM-BF4.
Nanomedical relevance of the intermolecular interaction dynamics - Examples from lysozymes and insulins Ruiyan Zhang, Ning Zhang, Marzieh Mohri, Lisha Wu, Thomas Eckert, Vadim B. Krylov, Andrea Antosova, Slavomira Ponikova, Zuzana Bednarikova, Philipp Markart, Andreas Günther, Bengt Norden, Martin Billeter, Roland Schauer, Axel J. Scheidig, Bhisma N. Ratha, Anirban Bhunia, Karsten Hesse, Mushira Abdelaziz Enani, Jürgen Steinmeyer, Athanasios K. Petridis, Tibor Kozar, Zuzana Gazova, Nikolay E. Nifantiev, Hans-Christian Siebert ACS Omega, 2019
Destroying activity of magnetoferritin on lysozyme amyloid fibrils Peter Kopcansky, Katarina Siposova, Lucia Melnikova, Zuzana Bednarikova, Milan Timko, Zuzana Mitroova, Andrea Antosova, Vasil M. Garamus, Viktor I. Petrenko, Mikhail V. Avdeev, Zuzana Gazova Journal of Magnetism and Magnetic Materials, 2015
