Catalytic nanomaterials, widely used as substitutes of peroxidase, exhibit unique properties, which are unattainable for native enzymes. However, their activity is usually examined by means of substrates developed and methods standardized for horseradish peroxidase (HRP). The aim of the presented work was to determine the scope of usefulness of chromogenic substrates for gold nanoparticle (AuNP) activity studies under conditions which significantly extend beyond the activity range of a native HRP. The applicability of chromogens such as 3,3′5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) beyond the typical range of pH, and for the samples of high concentration of hydrogen peroxide was examined. The conducted research confirmed the usefulness of ABTS and TMB in acidic media (pH 2.5–3.5). At the same time, potential interferences from chloride anion, unobservable for HRP-based assays, were indicated. Moreover, a number of potentially useful hints concerning relations of concentration of substrates and catalyst for aromatic amine oxidation (TMB and OPD) were proposed. By increasing the concentration of chromogens and thanks to assuring the relatively low conversion of the reaction, the stability of TMB and OPD oxidation product was improved even in acidic media. The comparative studies of H₂O₂ affinity to the surface of AuNPs in the presence of various hydrogen donors underlined the superiority of phenolic compounds over aromatic amines and ABTS in the case of the samples of relatively low H₂O₂ concentration. This work highlights some improvements in the methods of HRP-like activity characterization of NPs. It provides a critical analysis of the major challenges, which may emerge in a case of bioanalytical assays employing the catalytic nanoparticles as labels.

This article describes the work on synthesis, surface functionalization and characterization of hollow gold nanoshells (HGNs), and their application in a photothermal tumor therapy. The studied gold nanoshells were synthesized using a method based on a reduction of HAuCl4 onto silver nanoparticle templates. A selected aptamer - AS1411 - selective towards nucleolin, with a terminal thiol group was conjugated to HGNs. Surface functionalization of synthesized nanoparticles was necessary to gain the affinity to tumor cells, thus allowing the selective delivery of the modified nanoparticles to target cells. As expected, an increased selectivity of nanoconjugates towards tumor cells (A375) in a comparison to normal ones (HaCaT) was observed (2.67 times). In the framework of our studies the biological activity of the obtained nanoconjugates was evaluated using MTT assay. For this aim, both normal and tumor cell cultures respresenting skin tissue were exposed to gold nanoparticles solutions. It was found, that HGN-AS1411 conjugates cause less than 10% loss in a cell viability in the case of both cell lines at the highest tested concentration. The potential of the developed nanoconjugates as agents in PTT of skin cancer was investigated in a subsequent stage of our research. It was found, that A375 cell viability amounted to less than 40% for 75μM of nanoconjugates (expressed as a concentration of Au atoms) as a result of the laser irradiation of this cell culture after desired accumulation of the tested nanoconjugates inside A375 cells.

Nucleic acid extraction and purification are crucial steps in sample preparation for multiple diagnostic procedures. Routine methodologies of DNA isolation require benchtop equipment (e.g., centrifuges) and labor-intensive steps. Magnetic nanoparticles (MNPs) as solid-phase sorbents could simplify this procedure. A wide range of surface coatings employs various molecular interactions between dsDNA and magnetic nano-sorbents. However, a reliable, comparative evaluation of their performance is complex. In this work, selected Fe₃O₄ modifications, i.e., polyethyleneimine, gold, silica, and graphene derivatives, were comprehensively evaluated for applications in dsDNA extraction. A family of single batch nanoparticles was compared in terms of morphology (STEM), composition (ICP-MS/MS and elemental analysis), surface coating (UV-Vis, TGA, FTIR), and MNP charge (ζ-potential). ICP-MS/MS was also used to unify MNPs concentration allowing a reliable assessment of individual coatings on DNA extraction. Moreover, studies on adsorption medium (monovalent vs. divalent ions) and extraction buffer composition were carried out. As a result, essential relationships between nanoparticle coatings and DNA adsorption efficiencies have been noticed. Fe₃O₄@PEI MNPs turned out to be the most efficient nano sorbents. The optimized composition of the extraction buffer (medium containing 0.1 mM EDTA) helped avoid problems with Fe³⁺ stripping, which improved the validity of the spectroscopic determination of DNA recovery.

The aim of this work was to carry out comparative studies on the peroxidase-like activity of gold nanoparticles (AuNPs) stabilized with low molecular weight hyperbranched polyglycidol (HBPG-OH) and its derivative modified with maleic acid residues (HBPG-COOH). The influence of the stabilizer to gold precursor ratio on the size and morphology of nanoparticles obtained was checked, and prepared nanoparticles were characterized by means of transmission electron microscopy and UV-Vis spectroscopy. The results indicated the divergent effect of increasing the concentration of stabilizers (HBPG-OH or HBPG-COOH) on the size of the nanostructures obtained. The gold nanoparticles obtained were characterized as having intrinsic peroxidase-like activity and the mechanism of catalysis in acidic and alkaline mediums was consistent with the standard Michaelis–Menten kinetics, revealing a strong affinity of AuNPs with 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3, 3′, 5, 5′-tetramethylbenzidine (TMB), and significantly lower affinity towards phenol. By comparing the kinetic parameters, a negligible effect of polymeric ligand charge on activity against various types of substrates (anionic or cationic) was indicated. The superiority of steric stabilization via the application of tested low-weight hyperbranched polymers over typical stabilizers in preventing salt-induced aggregation and maintaining high catalytic activity in time was proved. The applied hyperbranched stabilizers provide a good tool for manufacturing gold-based nanozymes, which are highly stable and active over a wide pH range.

Reactive oxygen species (ROS) play an important role in various physiological processes of living organisms. However, their increased concentration is usually considered as a threat for our health. Plants, invertebrates, and vertebrates including humans have various enzymatic and non-enzymatic defence systems against ROS. Unfortunately, both bad condition of surrounding environment and unhealthy lifestyle can interfere with an activity of enzymes responsible for a regulation of ROS levels. Therefore, it is important to look for alternative ROS scavengers, which could be administrated to chosen tissues to prevent pathological processes such as distortion of DNA or RNA structures and oxidation of proteins and lipids. One of the most recently proposed solutions is the application of nanozymes, which could mimic the activity of essential enzymes and prevent excessive activity of ROS. In this work, nanoparticles of Au, Pt, Pd, Ru and Rh were synthesized and studied in this regard. Peroxidase-, catalase (CAT)- and superoxide dismutase (SOD)-like activity of obtained nanoparticles were tested and compared using different methods. The influence of bovine and human albumins on CAT- and peroxidase-like activity was examined. Moreover, in the case of CAT-like activity, an influence of pH and temperature was examined and compared. Determination of SOD-like activity using the methods described for the examination of the activity of native enzyme was not fully successful. Moreover, cytotoxicity of chosen nanoparticles was studied on both regular and tumor cells.