The first aim of this study was to fabricate vertically aligned multiwalled carbon nanotubes (MWCNTs). MWCNTs were successfully prepared by using plasma enhanced chemical vapour deposition. Further, three carbon composite electrodes with different content of carbon particles with various shapes and sizes were prepared and tested on measuring of nucleic acids. The dependences of adenine peak height on the concentration of nucleic acid sample were measured. Carbon composite electrode prepared from a mixture of glassy and spherical carbon powder and MWCNTs had the highest sensitivity to nucleic acids. Other interesting result is the fact that we were able to distinguish signals for all bases using this electrode.

• Publication type
• Journal Article MeSH

We used carbon paste electrodes and a standard potentiostat to detect silver ions. The detection limit (3 Signal/Noise ratio) was estimated as 0.5 μM. A standard electrochemical instrument microanalysis of silver(I) ions was suggested. As a working electrode a carbon tip (1 mL) or carbon pencil was used. Limits of detection estimated by dilution of a standard were 1 (carbon tip) or 10 nM (carbon pencil). Further we employed flow injection analysis coupled with carbon tip to detect silver(I) ions released in various beverages and mineral waters. During first, second and third week the amount of silver(I) ions releasing into water samples was under the detection limit of the technique used for their quantification. At the end of a thirteen weeks long experiment the content of silver(I) ions was several times higher compared to the beginning of release detected in the third week and was on the order of tens of nanomoles. In subsequent experiments the influence of silver(I) ions (0, 5 and 10 μM) on a plant model system (tobacco BY-2 cells) during a four-day exposition was investigated. Silver(I) ions were highly toxic to the cells, which was revealed by a double staining viability assay. Moreover we investigated the effect of silver(I) ions (0, 0.3, 0.6, 1.2 and 2.5 μM) on guppies (Poecilia reticulata). Content of Ag(I) increased with increasing time of the treatment and applied concentrations in fish tissues. It can be concluded that a carbon tip or carbon pencil coupled with a miniaturized potentiostat can be used for detection of silver(I) ions in environmental samples and thus represents a small, portable, low cost and easy-to-use instrument for such purposes.

• Keywords
• ecotoxicology, guppy (Poecilia reticulata), miniaturized carbon electrodes, silver, tobacco cells, voltammetry,
• Publication type
• Journal Article MeSH

Chloride ion sensing is important in many fields such as clinical diagnosis, environmental monitoring and industrial applications. We have measured chloride ions at a carbon paste electrode (CPE) and at a CPE modified with solid AgNO₃, a solution of AgNO₃ and/or solid silver particles. Detection limits (3 S/N) for chloride ions were 100 μM, 100 μM and 10 μM for solid AgNO₃, solution of AgNO₃ and/or solid silver particles, respectively. The CPE modified with silver particles is the most sensitive to the presence chloride ions. After that we approached to the miniaturization of the whole electrochemical instrument. Measurements were carried out on miniaturized instrument consisting of a potentiostat with dimensions 35 × 166 × 125 mm, screen printed electrodes, a peristaltic pump and a PC with control software. Under the most suitable experimental conditions (Britton-Robinson buffer, pH 1.8 and working electrode potential 550 mV) we estimated the limit of detection (3 S/N) as 500 nM.

• Keywords
• Amperometry, Carbon Paste Electrode, Chloride Ions, Screen Printed Electrode, Silver, Voltammetry,
• Publication type
• Journal Article MeSH

An Electrochemical Detection of Metallothioneins at the Zeptomole Level in Nanolitre VolumesWe report on improvement of the adsorptive transfer stripping technique (AdTS) coupled with the differential pulse voltammetry Brdicka reaction to determine a thiol-protein. The current technique has been unable to generate reproducible results when analyzing very low sample volumes (nanolitres). This obstacle can be overcome technically by modifying the current transfer technique including cooling step of the adsorbed analyte. We tested the technique on determination of a promising tumour disease marker protein called metallothionein (MT). The detection limit (3 S/N) of MT was evaluated as 500 zeptomoles per 500 nL (1 pM) and the quantification limit (10 S/N) as 1,500 zeptomoles per 500 nL (3 pM). Further, the improved AdTS technique was utilized to analyze blood serum samples from patients with breast cancer. Based on the results obtained it can be concluded that the improved technique can be used to detect a thiolprotein in very low sample volumes and can also prevent interferences during the washing and transferring step.

• Keywords
• Brdicka reaction, Proteomics, adsorptive transfer stripping technique, differential pulse voltammetry, human blood serum, metallothionein, thiols, tumour disease, zeptomole,
• Publication type
• Journal Article MeSH

Lactoferrin is a multifunctional protein with antimicrobial activity and others tohealth beneficial properties. The main aim of this work was to propose easy to usetechnique for lactoferrin isolation from cow colostrum samples. Primarily we utilizedsodium dodecyl sulphate - polyacrylamide gel electrophoresis for isolation of lactoferrinfrom the real samples. Moreover we tested automated microfluidic Experionelectrophoresis system to isolate lactoferrin from the collostrum sample. The welldeveloped signal of lactoferrin was determined with detection limit (3 S/N) of 20 ng/ml. Inspite of the fact that Experion is faster than SDS-PAGE both separation techniques cannotbe used in routine analysis. Therefore we have tested third separation technique, ionexchange chromatography, using monolithic column coupled with UV-VIS detector (LCUV-VIS). We optimized wave length (280 nm), ionic strength of the elution solution (1.5M NaCl) and flow rate of the retention and elution solutions (0.25 ml/min and 0.75 ml/min.respectively). Under the optimal conditions the detection limit was estimated as 0.1 μg/mlof lactoferrin measured. Using LC-UV-VIS we determined that lactoferrin concentrationvaried from 0.5 g/l to 1.1 g/l in cow colostrums collected in the certain time interval up to 72 hours after birth. Further we focused on miniaturization of detection device. We testedamperometric detection at carbon electrode. The results encouraged us to attempt tominiaturise whole detection system and to test it on analysis of real samples of humanfaeces, because lactoferrin level in faeces is closely associated with the inflammations ofintestine mucous membrane. For the purpose of miniaturization we employed thetechnology of printed electrodes. The detection limit of lactoferrin was estimated as 10μg/ml measured by the screen-printed electrodes fabricated by us. The fabricatedelectrodes were compared with commercially available ones. It follows from the obtainedresults that the responses measured by commercial electrodes are app. ten times highercompared with those measured by the electrodes fabricated by us. This phenomenonrelates with smaller working electrode surface area of the electrodes fabricated by us(about 50 %) compared to the commercial ones. The screen-printed electrodes fabricatedby us were utilized for determination of lactoferrin faeces. Regarding to fact that sample offaeces was obtained from young and healthy man the amount of lactoferrin in sample wasunder the limit of detection of this method.

• Keywords
• Electrochemistry, Lactoferrin, Linear Sweep Voltammetry, Liquid Chromatography, Milk Protein, Monolithic Column, Screen-Printed Carbon Electrode, UV-VIS Spectrometry,
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

About biological affecting of flavonoids on animal organisms is known less,thus we selected flavonoids, flavanones and flavones, and their glycosides, which wereexamined as potential inducers of cytochrome(s) P450 when administrated by gavages intoexperimental male rats. The study was focused on induction of CYP1A1, the majorcytochrome P450 involved in carcinogen activation. The data obtained demonstrate thenecessity of taking into account not only ability of flavonoids to bind to Ah receptor(induction factor) but also to concentrate on their distribution and metabolism (includingcolon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode. Primarily we aimed on investigation of their basic electrochemical behaviour. After that we have optimized frequency, step potential and supporting electrolyte. Based on the results obtained, we selected the most suitable conditions for determination of the flavonoids as follows: frequency 180 Hz, step potential 1.95 mV/s and phosphate buffer of pH 7 as supporting electrolyte. Detection limits (3 S/N) of the flavonoids were from units to tens of nM except diosmin, where the limit were higher than μM. In addition, we attempted to suggest a sensor for analysis of flavonoids in urine. It clearly follows from the results obtained that flavonoids can be analysed in the presence of animal urine, because urine did not influence much the signals of flavonoids (recoveries of the signals were about 90 %).

• Keywords
• antioxidant, cancer, carbon paste electrode, cytochrome P450, flavonoids, square wave voltammetry, western blot analysis,
• Publication type
• Journal Article MeSH

Presence of mutated and/or structurally modified (e.g., denatured, aggregated) protein p53 form is associated with several disorders such as Alzheimer's disease, Parkinson's disease, prion diseases, and many types of tumours. The aim of this work was to distinguish native, denatured and aggregated form of full-length p53 by flow injection analysis coupled with electrochemical detector (FIA-ED). Firstly FIA-ED method used for protein native form determination was optimized (detection limit 45.8 amol per 5 mul injection; 3 x S/N). In addition the technique was applied to identify p53 structural forms (denatured and aggregated). It was found out that denatured form provides about three times higher electrochemical response (protein structure unfolding, approach of more electroactive centers - aminoacid residues - towards electrode surface) in comparison with native form. On the other hand, aggregated form offers lower response (steric eclipse of electroactive protein parts) when compared with the signal of native form. The obtained data show that we are not only able to sensitively determine native, denatured, and aggregated structural forms of p53 protein but also to distinguish them.

• MeSH
• Protein Denaturation MeSH
• Electrochemistry MeSH
• Protein Conformation MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   MeSH
• Flow Injection Analysis methods   MeSH
• Sensitivity and Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH