DNA-based molecular recognition system for lactoferrin biosensing.

The paper describes the development of a novel DNA oligonucleotide-based affinity bioreceptor that binds to lactoferrin, a glycoprotein-type immunomodulator. The research was performed using surface plasmon resonance method to investigate affinity of various types of oligonucleotides to the target protein. The 72 base pair-long 5'[(TAGAGGATCAAA)AAA]4TAGAGGATCAAA3' sequence with the highest affinity to lactoferrin was selected for further investigations. Kinetic analysis of the interaction between selected DNA and lactoferrin provided rate and equilibrium constants: ka = (2.49 ± 0.03)∙104 M-1∙s-1, kd = (1.89 ± 0.02)∙10-3 s-1, KA = (0.13 ± 0.05)∙108 M-1, and KD = (7.61 ± 0.18)∙10-8 M. Thermodynamic study conducted to determine the ΔH0, ΔS0, and ΔG0 for van't Hoff characteristic in the temperature range of 291.15-305.15 K, revealed the complex formation as endothermic and entropically driven. The chosen DNA sequence's selectivity towards lactoferrin was confirmed with interferents' response constituting <3 % of the response to lactoferrin. SPR analysis justified utility of the designed DNA oligonucleotide for Lf determination, with LOD of 4.42∙10-9 M. Finally, the interaction between lactoferrin and DNA was confirmed by electrochemical impedance spectroscopy, providing the basis for further quantitative assay of lactoferrin using the developed DNA-based bioreceptor. The interactions were performed under immobilized DNA ligand conditions, thus reflecting the sensor's surface, which facilitates their transfer to other label-free biosensor technologies.

Electrodes Based on a Titanium Dioxide Nanotube-Spherical Silver Nanoparticle Composite for Sensing of Proteins.

The aim of the research was to provide electrochemical, chemical, phase, and microscopic characteristics of electrodes based on titanium dioxide nanotubes (TNTs) containing uniformly deposited, nonagglomerated spherical silver nanoparticles (AgNPs). The nanoparticles were produced with the use of electrodeposition and sputter deposition methods. This paper presents the results of research of these platforms with the use of the following techniques: electrochemical impedance spectroscopy, X-ray diffraction analysis, X-ray photoelectron spectroscopy, and scanning electron microscopy. Evaluation of the adsorption of proteins-bovine serum albumin (BSA)-was carried out to establish the possibility of the use of the electrodes in a low-cost, simple detection system without surface functionalization. The research proved that the AgNP deposition facilitated the electron transfer increasing their conductivity properties as well as promoting the protein adsorption. The AgNPs/TNT electrodes showed a high selectivity to the BSA-anti-BSA complex. Half an hour of immobilization was enough to completely saturate the TNT electrodes, whereas for AgNPs/TNTs, 1 h of immobilization seemed to be not enough. The impedance parameter changes for electrodes with the AgNPs reached even about 300%. The biggest changes were noted for the platform obtained using cyclic voltammetry, so it is the best detection platform for biosensing.

Influence of geometry and annealing temperature in argon atmosphere of TiO2 nanotubes on their electrochemical properties.

PURPOSE: In this paper, electrochemical properties of the as-formed and thermally treated titanium dioxide (TiO2) nanotubes with diameter in the range of 20-100 nm and height in the range of 100-1000 nm were presented. In addition, the effects of annealing temperature (450-550 °C) on the electrochemical characteristics of these structures, as well as the influence of diameter and height of TiO2 nanotubes on these properties were examined. The results were referred to a compact TiO2 layer (100 nm thick). METHODS: The electrochemical test included open circuit potential, impedance spectroscopy and cyclic voltammetry measurements. The scanning electron microscope with energy dispersive spectroscopy analyser, x-ray photoelectron spectroscopy, and x-ray diffraction analysers were used for surface morphology characterisation as well as elemental, phase and chemical composition of TiO2 layers. RESULTS: It was found that nanotubes with the diameter of 50 and 75 nm (height of 1000 nm) annealed at 550 °C exhibit the lowest impedance and phase angle values. However, the voltammetric detection of potassium ferricyanide indicated that the closest to 1 Ipc /Ipa ratio were shown by nanotubes with a diameter of 50 and 75 nm annealed at 450 °C. CONCLUSIONS: On the basis of performed analysis, it can be stated that the TiO2 layer with nanotubes of 50 nm in diameter and of 1000 nm in height, annealed in 450 °C may be indicated as the ones having the most favourable sensing and biosensing properties.

Influence of the Silver Nanoparticles (AgNPs) Formation Conditions onto Titanium Dioxide (TiO2) Nanotubes Based Electrodes on Their Impedimetric Response.

This paper presents the comparison of the effects of three methods of production of silver spherical and near-spherical nanoparticles (AgNPs) on the titanium dioxide nanotubes (TNT) base: cyclic voltammetry, chronoamperometry, and sputter deposition. It also evaluates the influence of silver nanoparticles on the electrochemical properties of the developed electrodes. The novelty of this research was to fabricate regular AgNPs free of agglomerates uniformly distributed onto the TNT layer, which has not been accomplished with previous attempts. The applied methods do not require stabilizing and reducing reagents. The extensive electrochemical characteristic of AgNP/TNT was performed by open circuit potential and electrochemical impedance spectroscopy methods. For AgNPs/TNT obtained by each method, the impedance module of these electrodes was up to 50% lower when compared to TNT, which means that AgNPs enabled more efficient electron transfer due to the effective area increase. In addition, the presence of nanoparticles increases the corrosion resistance of the prepared electrodes. These substrates can be used as electrochemical sensors due to their high electrical conductivity, and also as implants due to the antibacterial properties of both the TNT and AgNPs.

Liver tissue fragments obtained from males are the most promising source of human hepatocytes for cell-based therapies - Flow cytometric analysis of albumin expression.

Cell-based therapies that could provide an alternative treatment for the end-stage liver disease require an adequate source of functional hepatocytes. There is little scientific evidence for the influence of patient's age, sex, and chemotherapy on the cell isolation efficiency and metabolic activity of the harvested hepatocytes. The purpose of this study was to investigate whether hepatocytes derived from different sources display differential viability and biosynthetic capacity. Liver cells were isolated from 41 different human tissue specimens. Hepatocytes were labeled using specific antibodies and analyzed using flow cytometry. Multiparametric analysis of the acquired data revealed statistically significant differences between some studied groups of patients. Generally, populations of cells isolated from the male specimens had greater percentage of biosynthetically active hepatocytes than those from the female ones regardless of age and previous chemotherapy of the patient. Based on the albumin staining (and partially on the α-1-antitrypsin labeling) after donor liver exclusion (6 out of 41 samples), our results indicated that: 1. samples obtained from males gave a greater percentage of active hepatocytes than those from females (p = 0.034), and 2. specimens from the males after chemotherapy greater than those from the treated females (p = 0.032).

Dried human skin fibroblasts as a new substratum for functional culture of hepatic cells.

The primary hepatocytes culture is still one of the main challenges in toxicology studies in the drug discovery process, development of in vitro models to study liver function, and cell-based therapies. Isolated hepatocytes display a rapid decline in viability and liver-specific functions including albumin production, conversion of ammonia to urea, and activity of the drug metabolizing enzymes. A number of methods have been developed in order to maintain hepatocytes in their highly differentiated state in vitro. Optimization of culture conditions includes a variety of media formulations and supplements, growth surface coating with the components of extracellular matrix or with synthetic polymers, three-dimensional growth scaffolds and decellularized tissues, and coculture with other cell types required for the normal cell-cell interactions. Here we propose a new substratum for hepatic cells made by drying confluent human skin fibroblasts' culture. This growth surface coating, prepared using maximally simplified procedure, combines the advantages of the use of extracellular matrices and growth factors/cytokines secreted by the feeder layer cells. In comparison to the hepatoma cells grown on a regular tissue culture plastic, cells cultured on the dried fibroblasts were able to synthesize albumin in larger quantities and to form greater number of apical vacuoles. Unlike the coculture with the living feeder layer cells, the number of cells grown on the new substratum was not reduced after fourteen days of culture. This fact could make the dried fibroblasts coating an ideal candidate for the substrate for non-dividing human hepatocytes.

Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines.

Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests.

Evaluation of the effects of antibiotics on cytotoxicity of EGFP and DsRed2 fluorescent proteins used for stable cell labeling.

The use of fluorescent markers has proven to be an attractive tool in biological imaging. However, its usefulness may be confined by the cytotoxicity of the fluorescent proteins. In this article, for the first time, we have examined an influence of the antibiotics present in culture medium on cytotoxicity of the EGFP and DsRed2 markers used for whole-cell labeling. Results showed that doxycycline negatively affected albumin synthesis in DsRed2-expressing hepatoma cells, and that both hepatoma cells and human skin fibroblasts, labeled with this protein, were characterized by the lowered growth rates. Thus, the cytotoxic effect of fluorescent markers depends on both protein used for cell labeling and on growth conditions that may cause cell stress.