Kristyna Smerkova

Investigation of interaction between magnetic silica particles and lambda phage DNA fragment

Nucleic acids belong to the most important molecules and therefore the understanding of their properties, function and behavior is crucial. Even though a range of analytical and biochemical methods have been developed for this purpose, one common step is essential for all of them - isolation of the nucleic acid from the from complex sample matrix. The use of magnetic particles for the separation of nucleic acids has many advantages over other isolation methods. In this study, an isolation procedure for extraction of DNA was optimized. Each step of the isolation process including washing, immobilization and elution was optimized and therefore the efficiency was increased from 1.7% to 28.7% and the total time was shortened from 75 to 30min comparing to the previously described method. Quantification of the particular parameter influence was performed by square-wave voltammetry using hanging drop mercury electrode. Further, we compared the optimized method with standard chloroform extraction and applied on isolation of DNA from Staphylococcus aureus and Escherichia coli.

DNA interaction with zinc(II) ions

We focused on interactions of Zn(II) with DNA in this study. These interactions were monitored using UV/vis spectrophotometry and gel electrophoresis. Firstly, we isolated and amplified 498 bp fragment of DNA. Samples were obtained by incubation of DNA fragment with Zn(II) for 60 min at 25 °C. After incubation, the samples were dialyzed and analyzed immediately. In this way, DNA was converted into a metal bound DNA (Zn-DNA). Interaction of Zn(II) with DNA caused change in the absorption spectrum (190-350 nm) and decrease in the melting temperature (Tm) of Zn-DNA. Spectrophotometric (UV/vis) analysis showed that increasing concentrations of zinc(II) ions led to the increase in the absorbance at 200 nm and decrease in absorbance at 251 nm. Application of zinc(II) ions at 5.5 μM concentration caused decrease in Tm for app. 7.5 °C in average in comparison with control (75.5 ± 3 °C). The lowest melting temperature (60.5 ± 2.5 °C) was observed after application of zinc(II) ions at 33 μM concentration. Gel electrophoresis proved significance of Zn(II) in the renaturation of DNA. Samples of Zn-DNA (15 μM DNA+5.5-55 μM Zn(II)) caused significant changes in the renaturation of DNA in comparison with the control, untreated DNA (15 μM DNA).

Integrated chip electrophoresis and magnetic particle isolation used for detection of hepatitis B virus oligonucleotides

Rapid and sensitive detection is a key step in the effective and early response to the global hazard of various viral diseases. In this study, an integrated isolation of hepatitis B virus (HBV)‐specific DNA fragment by magnetic nanoparticles (MNPs) and its immediate analysis by microchip CGE was performed. Microfluidic CE chip was used to accommodate the complete process of viral DNA isolation by MNPs including hybridization and thermal denaturation followed by CE separation. Beforehand, calibration curves of HBV fragments were constructed. For isolation by MNPs, specific streptavidin–biotin interaction was used to bind complementary HBV fragment to magnetic particles. After analysis of isolated HBV by regular MNPs method, innovative approach was performed. The commercial CE chip (Bio‐rad) was successfully used to execute HBV fragment isolation. Detection using LIF with detection limit of 1 ng/mL was accomplished.

Glutathione modified CdTe quantum dots as a label for studying DNA interactions with platinum based cytostatics

Cisplatin, carboplatin, and oxaliplatin represent three generations of platinum based drugs applied successfully for cancer treatment. As a consequence of the employment of platinum based cytostatics in the cancer treatment, it became necessary to study the mechanism of their action. Current accepted opinion is the formation of Pt‐DNA adducts, but the mechanism of their formation is still unclear. Nanomaterials, as a progressively developing branch, can offer a tool for studying the interactions of these drugs with DNA. In this study, fluorescent CdTe quantum dots (QDs, λem = 525 nm) were employed to investigate the interactions of platinum cytostatics (cisplatin, carboplatin, and oxaliplatin) with DNA fragment (500 bp, c = 25 μg/mL). Primarily, the fluorescent behavior of QDs in the presence of platinum cytostatics was monitored and major differences in the interaction of QDs with tested drugs were observed. It was found that the presence of carboplatin (c = 0.25 mg/mL) had no significant influence on QDs fluorescence; however cisplatin and oxaliplatin quenched the fluorescence significantly (average decrease of 20%) at the same concentration. Subsequently, the amount of platinum incorporated in DNA was determined by QDs fluorescence quenching. Best results were reached using oxaliplatin (9.4% quenching). Linear trend (R2 = 0.9811) was observed for DNA platinated by three different concentrations of oxaliplatin (0.250, 0.125, and 0.063 mg/mL). Correlation with differential pulse voltammetric measurements provided linear trend (R2 = 0.9511). As a conclusion, especially in the case of oxaliplatin‐DNA adducts, the quenching was the most significant compared to cisplatin and nonquenching carboplatin.

Platinum nanoparticles induce damage to DNA and inhibit DNA replication

Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8–11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent.

Study of Linkage between Glutathione Pathway and the Antibiotic Resistance of Escherichia coli from Patients' Swabs

In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase—GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (Km 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of “more resistant” E. coli exhibited differences in Km between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group.

Label-free and amplification-free miR-124 detection in human cells

MicroRNAs (miRNAs) are becoming a very important group of molecules especially since their connection to numerous diseases has been revealed. The potential in gene therapy as well as in diagnostics is being widely investigated leading to the demand of sensitive, selective and simple methods of isolation and detection. The combined advantages of magnetic particle-based separation with sensitive electrochemical detection may offer a very valuable tool for these purposes. In this study, the miR‑124 was targeted as an example analyte for development and optimization of the isolation procedure coupled to the electrochemical detection. The sensitivity of the method was demonstrated by the limit of detection at the level of nanomolar concentration (4 nM). To verify the applicability of the procedure to the real samples, miR‑124 was isolated from the human embryonic kidney cells naturally expressing this miRNA molecule and the results were compared to the amount of miR‑124 isolated from the cells transfected by the pENTR-miR‑124 plasmid leading to the overexpression of miR‑124.

Short-sweep capillary electrophoresis with a selective zinc fluorescence imaging reagent FluoZin-3 for determination of free and metalothionein-2a-bound Zn 2+ ions

A capillary electrophoretic (CE) method using a short-sweep approach and laser-induced fluorescence (LIF) detection (ShortSweepCE-LIF) was developed for determination of Zn2+ and Cd2+ as complexes with highly selective and sensitive fluorescent probe FluoZin-3. The ShortSweepCE-LIF method, established in this work, can be used for examining competitive Zn2+ and Cd2+ binding properties of metalloproteins or peptides. The parameters including background electrolyte composition, injection pressure and time as well as separation voltage were investigated. Under the optimized conditions, 80 mM HEPES, pH 7.4, with 1.5 μM FluoZin-3 was used as an electrolyte, hydrodynamic injection was performed at 50 mbar for 5 s, and separation voltage of 25 kV. Limits of detection for Zn2+ and Cd2+ were 4 and 125 nM, respectively. The developed method was demonstrated in a study of interactions between metalothionein-2a isoform and metal ions Zn2+, Co2+ and Cd2+. It was found that FluoZin-3 was able to extract a single Zn2+ ion, while added Co2+ (in surplus) extracted only 2.4 Zn2+ ions, and Cd2+ extracted all 7 Zn2+ ions present in the metalothionein molecule.

Doxorubicin interactions with bovine serum albumin revealed by microdialysis with on‐line laser‐induced fluorescence detection at subpicogram level

Doxorubicin (DOX) is an effective antitumor drug employed for treatment of a wide range of cancers types such as neuroblastoma, osteosarcoma, breast and esophageal carcinomas. On the other hand, the cumulative dose is restricted (300–550 mg/m2) and its amount administered to a patient has to be closely controlled due to its cardiotoxicity. To understand the mechanisms of the DOX side effects as well as to reveal the ways how to reduce its adverse impact on cardiomyocytes, the interactions with particular components of the blood and tissues have to be studied in greater detail. In this work, microdialysis technique was optimized to extract DOX from samples and subsequently monitor its interaction with BSA. Finally, the microdialysis probe was connected on‐line to the LIF detector to ensure the real‐time detection. The best flow rate was 1 μL/min and after 120 min of microdialysis 28% of the DOX was dialyzed out from the sample. The results from investigation of the DOX–BSA interaction indicate that the interaction occurs in less than 30 min, causing marked decrease in the amount of DOX extracted by microdialysis.

Utilization of paramagnetic microparticles for automated isolation of free circulating mRNA as a new tool in prostate cancer diagnostics

Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin‐modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20, serum vs. oligo(dT)20, serum and SMPs). RNA content was measured, and the expression of metallothionein‐2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT‐PCR detection. Isolation is possible on serum volume range (10–200 μL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 μL, with optimal results within 10–30 μL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1–0.4 μL. This optimized protocol was also modified to fit needs of automated one‐step single‐tube analysis with identical efficiency compared to conventional setup. One‐step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process.