Marketa Ryvolova

Fully automated spectrometric protocols for determination of antioxidant activity: advantages and disadvantages.

The aim of this study was to describe behaviour, kinetics, time courses and limitations of the six different fully automated spectrometric methods - DPPH, TEAC, FRAP, DMPD, Free Radicals and Blue CrO5. Absorption curves were measured and absorbance maxima were found. All methods were calibrated using the standard compounds Trolox® and/or gallic acid. Calibration curves were determined (relative standard deviation was within the range from 1.5 to 2.5 %). The obtained characteristics were compared and discussed. Moreover, the data obtained were applied to optimize and to automate all mentioned protocols. Automatic analyzer allowed us to analyse simultaneously larger set of samples, to decrease the measurement time, to eliminate the errors and to provide data of higher quality in comparison to manual analysis. The total time of analysis for one sample was decreased to 10 min for all six methods. In contrary, the total time of manual spectrometric determination was approximately 120 min. The obtained data provided good correlations between studied methods (R = 0.97 – 0.99).

Nanocarriers for anticancer drugs--new trends in nanomedicine.

This review provides a brief overview of the variety of carriers employed for targeted drug delivery used in cancer therapy and summarizes advantages and disadvantages of each approach. Particularly, the attention was paid to polymeric nanocarriers, liposomes, micelles, polyethylene glycol, poly(lactic-co-glycolic acid), dendrimers, gold and magnetic nanoparticles, quantum dots, silica nanoparticles, and carbon nanotubes. Further, this paper briefly focuses on several anticancer agents (paclitaxel, docetaxel, camptothecin, doxorubicin, daunorubicin, cisplatin, curcumin, and geldanamycin) and on the influence of their combination with nanoparticulate transporters to their properties such as cytotoxicity, short life time and/or solubility.

Metallothioneins and zinc in cancer diagnosis and therapy.

Metallothioneins (MTs) are involved in protection against oxidative stress (OS) and toxic metals and they participate in zinc metabolism and its homeostasis. Disturbing of zinc homeostasis can lead to formation of reactive oxygen species, which can result in OS causing alterations in immunity, aging, and civilization diseases, but also in cancer development. It is not surprising that altered zinc metabolism and expression of MTs are of great interest in the case of studying of oncogenesis and cancer prognosis. The role of MTs and zinc in cancer development is tightly connected, and the structure and function of MTs are strongly dependent on Zn2+ redox state and its binding to proteins. Antiapoptic effects of MTs and their interactions with proteins nuclear factor kappa B, protein kinase C, esophageal cancer-related gene, and p53 as well as the role of MTs in their proliferation, immunomodulation, enzyme activation, and interaction with nitric oxide are reviewed. Utilization of MTs in cancer diagnosis and therapy is summarized and their importance for chemoresistance is also mentioned.

Modern Micro and Nanoparticle-Based Imaging Techniques

The requirements for early diagnostics as well as effective treatment of insidious diseases such as cancer constantly increase the pressure on development of efficient and reliable methods for targeted drug/gene delivery as well as imaging of the treatment success/failure. One of the most recent approaches covering both the drug delivery as well as the imaging aspects is benefitting from the unique properties of nanomaterials. Therefore a new field called nanomedicine is attracting continuously growing attention. Nanoparticles, including fluorescent semiconductor nanocrystals (quantum dots) and magnetic nanoparticles, have proven their excellent properties for in vivo imaging techniques in a number of modalities such as magnetic resonance and fluorescence imaging, respectively. In this article, we review the main properties and applications of nanoparticles in various in vitro imaging techniques, including microscopy and/or laser breakdown spectroscopy and in vivo methods such as magnetic resonance imaging and/or fluorescence-based imaging. Moreover the advantages of the drug delivery performed by nanocarriers such as iron oxides, gold, biodegradable polymers, dendrimers, lipid based carriers such as liposomes or micelles are also highlighted.

Biotin‐modified glutathione as a functionalized coating for bioconjugation of CdTe‐based quantum dots

In this study, biotin‐conjugated glutathione was synthesized using peptide bonding of the biotin carboxy group and amino group of the γ‐glutamic acid to prepare an alternative coating for CdTe quantum dots (QDs). This type of coating combines the functionality of the biotin with the fluorescent properties of the QDs to create a specific, high‐affinity fluorescent probe able to react with avidin, streptavidin and/or neutravidin. Biotin‐functionalized glutathione‐coated CdTe QDs were prepared by a simple one‐step method using Na2TeO3 and CdCl2. Obtained QDs were separated from the excess of the biotin‐conjugated glutathione by CE employing 300 mM borate buffer with pH 7.8 as a background electrolyte. The detection of sample components was performed by the photometric detection at 214 nm and LIF employing Ar+ ion laser (488 nm).

Paramagnetic particles coupled with an automated flow injection analysis as a tool for influenza viral protein detection.

Currently, the influenza virus infects millions of individuals every year. Since the influenza virus represents one of the greatest threats, it is necessary to develop a diagnostic technique that can quickly, inexpensively, and accurately detect the virus to effectively treat and control seasonal and pandemic strains. This study presents an alternative to current detection methods. The flow‐injection analysis‐based biosensor, which can rapidly and economically analyze a wide panel of influenza virus strains by using paramagnetic particles modified with glycan, can selectively bind to specific viral A/H5N1/Vietnam/1203/2004 protein‐labeled quantum dots. Optimized detection of cadmium sulfide quantum dots (CdS QDs)‐protein complexes connected to paramagnetic microbeads was performed using differential pulse voltammetry on the surface of a hanging mercury drop electrode (HMDE) and/or glassy carbon electrode (GCE). Detection limit (3 S/N) estimations based on cadmium(II) ions quantification were 0.1 μg/mL or 10 μg/mL viral protein at HMDE or GCE, respectively. Viral protein detection was directly determined using differential pulse voltammetry Brdicka reaction. The limit detection (3 S/N) of viral protein was estimated as 0.1 μg/mL. Streptavidin‐modified paramagnetic particles were mixed with biotinylated selective glycan to modify their surfaces. Under optimized conditions (250 μg/mL of glycan, 30‐min long interaction with viral protein, 25°C and 400 rpm), the viral protein labeled with quantum dots was selectively isolated and its cadmium(II) content was determined. Cadmium was present in detectable amounts of 10 ng per mg of protein. Using this method, submicrogram concentrations of viral proteins can be identified.

Electrophoretic fingerprint metallothionein analysis as a potential prostate cancer biomarker.

Prostate‐specific antigen (PSA) is a routinely used marker of prostate cancer; however, the cut‐off values for unambiguous positive/negative prostate cancer diagnoses are not defined. Therefore, despite the best effort, certain percentage of misdiagnosed cases is being recorded every year. For this reason, search for more specific diagnostic markers is of great interest. In this study, systematic comparison of PSA and metallothionein (MT) levels in blood serum of 46 prostate cancer‐diagnosed patients is presented. It is clearly demonstrated that PSA levels vary significantly and despite normal total PSA values in the range of 0 – 4 ng/mL were obtained in over 36.9% of cases, positive prostate cancer was diagnosed by biopsy. In contrary, MT levels were considerably elevated in all tested samples and no significant variations were observed. These results are indicating the potential of MT as an additional prostate cancer marker reducing, in combination with PSA, the probability of false positive/negative diagnosis. To increase the throughput of the screening, chip‐based capillary electrophoresis was suggested as a rapid and effective method for the fingerprinting analysis of prostate cancer from diseased blood sera.

Tissue Specific Electrochemical Fingerprinting

Background Proteomics and metalloproteomics are rapidly developing interdisciplinary fields providing enormous amounts of data to be classified, evaluated and interpreted. Approaches offered by bioinformatics and also by biostatistical data analysis and treatment are therefore of extreme interest. Numerous methods are now available as commercial or open source tools for data processing and modelling ready to support the analysis of various datasets. The analysis of scientific data remains a big challenge, because each new task sets its specific requirements and constraints that call for the design of a targeted data pre-processing approach. Methodology/Principal Findings This study proposes a mathematical approach for evaluating and classifying datasets obtained by electrochemical analysis of metallothionein in rat 9 tissues (brain, heart, kidney, eye, spleen, gonad, blood, liver and femoral muscle). Tissue extracts were heated and then analysed using the differential pulse voltammetry Brdicka reaction. The voltammograms were subsequently processed. Classification models were designed making separate use of two groups of attributes, namely attributes describing local extremes, and derived attributes resulting from the level = 5 wavelet transform. Conclusions/Significance On the basis of our results, we were able to construct a decision tree that makes it possible to distinguish among electrochemical analysis data resulting from measurements of all the considered tissues. In other words, we found a way to classify an unknown rat tissue based on electrochemical analysis of the metallothionein in this tissue.

Immunoextraction of zinc proteins from human plasma using chicken yolk antibodies immobilized onto paramagnetic particles and their electrophoretic analysis.

Zinc(II) as the only transition metal lacking redox activity is an essential part of approximately 10% proteins as a cofactor of these proteins. Considering the fact that there are numerous zinc(II) containing proteins, proteomics and metallomics studies aimed on them require accurate methods for preparation of real biological samples prior to their subsequent analysis using 2DE and MS. For this purpose, we suggested a new method based on chicken anti‐zinc antibodies and magnetizable particles. Antibodies were covalently immobilized to the surface of paramagnetic beads activated with tosyl group. Binding of the antibody to the beads was confirmed by secondary anti‐chicken antibody conjugated with horseradish peroxidase. The immunoextraction conditions, such as concentration of the beads (6–18 μg/mL of the sample), time of immunoextraction (6–34 min), pH and composition of the elution buffer, and time of extraction (48–300 s) were optimized. Subsequently, zinc proteins were extracted from human plasma and total concentration of zinc was monitored by electrochemical detection in the extracts. Under optimal conditions it was possible to monitor the proteins and zinc removal from the sample by chip CE, SDS‐PAGE, and indirectly using electrochemistry.

From amino acids to proteins as targets for metal-based drugs.

Metallomics and metalloproteomics are emerging fields addressing the role, uptake, transport and storage of trace metals ions both toxic and essential for an organism. Research areas related to the understanding of the mechanisms of life processes associated to metals are covered. Similarly to the genome and proteome terms, metallome was introduced to refer to metalloproteins, metalloenzymes and other metal-containing biomolecules in a biological system. This review aims to give an overview of metal ions behaviour in organisms. The interactions of metals with biomolecules such as amino acids, peptides and protein are the main focus. Special attention is paid to the application of nanotechnology-based techniques using these interactions for medical purposes such as diagnostics, imaging and therapy.