Study of Streptavidin-Modified Quantum Dots by Capillary Electrophoresis

Great boom of nanotechnologies impacts almost all areas of science and therefore detail understanding of the properties of nanomaterials as well as their interaction abilities is required. Surface modification and functionalization of nanoparticles is of a great interest due to the wide range of applications in the area of nanomedicine, nanobiology, and/or biochemistry. In this study, CdTe QDs were synthesized using microwave reactor and their surface was modified by streptavidin to ensure further suitability for bioconjugation with biotin-labelled oligonucleotides. For characterization of the synthesized QDs and for monitoring of the interaction with the oligonucleotide, capillary and gel electrophoresis was used. Moreover, complementary advantages of absorption (CE–UV) and laser-induced fluorescence detection (CE–LIF) were exploited. Comparison the electrophoretic mobilities obtained for streptavidin-modified QDs by CE–LIF (?9.87 × 10^?9 m²/V/s) and by CE–UV (?10.02 × 10^?9 m²/V/s) was in a good agreement enabling us to identify the peak of streptavidin-modified QDs in the CE–UV electropherogram containing also the peak of unreacted streptavidin. Subsequent conjugation of streptavidin-modified QDs with two model biotinylated oligonucleotides (BCL-2 and HBV) led to formation of the complex represented in the electropherograms as a very sharp peak. This peak height increased with time for 15.5 and 27 mAU using BCL-2 oligonucleotide and HBV oligonucleotide, respectively during 30 min interaction.     

Rapid superparamagnetic‐beads‐based automated immunoseparation of Zn‐proteins from Staphylococcus aureus with nanogram yield

Pathogenic bacteria have become a serious socio‐economic concern. Immunomagnetic separation‐based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles‐based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn‐proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn‐proteins by superparamagnetic beads modified with chicken anti‐Zn(II) antibody. S. aureus culture was treated with ZnCl₂. Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle–surface interactions were tested and associated protein profiles were identified using SDS‐PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn‐protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS‐PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.     

Microfluidic tool based on the antibody-modified paramagnetic particles for detection of 8-hydroxy-2'-deoxyguanosine in urine of prostate cancer patients

Guanosine derivatives are important for diagnosis of oxidative DNA damage including 8-hydroxy-2'-deoxyguanosine (8-OHdG) as one of the most abundant products of DNA oxidation. This compound is commonly determined in urine, which makes 8-OHdG a good non-invasive marker of oxidation stress. In this study, we optimized and tested the isolation of 8-OHdG from biological matrix by using paramagnetic particles with an antibody-modified surface. 8-OHdG was determined using 1-naphthol generated by alkaline phosphatase conjugated with the secondary antibody. 1-Naphthol was determined by stopped flow injection analysis (SFIA) with electrochemical detector using a glassy carbon working electrode and by stationary electrochemical detection using linear sweep voltammetry. A special modular electrochemical SFIA system which needs only 10 μL of sample including working buffer for one analysis was completely designed and successfully verified. The recoveries in different matrices and analyte concentration were estimated. Detection limit (3 S/N) was estimated as 5 pg/mL of 8-OHdG. This method promises to be very easily modified to microfluidic systems as "lab on valve". The optimized method had sufficient selectivity and thus could be used for determination of 8-OHDG in human urine and therefore for estimation of oxidative DNA damage as a result of oxidation stress in prostate cancer patients.     

Considering multiple occupancy of cavities in clathrate hydrate phase equilibrium calculations

One of the major assumptions of the original van der Waals–Platteeuw (vdWP) model is the single occupancy of hydrate cavities. In this work, the vdWP model is modified to also account for multiple occupancies of hydrate cavities by small molecules. The developed model is evaluated by calculating the hydrate equilibrium conditions with either oxygen or nitrogen as guest molecules in pure form, as well as mixtures of nitrogen and propane (molecules of these pure gases and those in (nitrogen + propane) have double occupancy in large cavities of structure II up to a certain concentration of propane). The results of this modified model show good agreement with the experimental data reported in the literature.     

Chip-Based CE for Avidin Determination in Transgenic Tobacco and Its Comparison with Square-Wave Voltammetry and Standard Gel Electrophoresis

Avidin transgenic plants are a potential tool for providing resistance against various species of insect pests due to the sequestration of vitamin H (biotin) in the plant from the insect pests. In this project we compared three techniques for avidin determination in transgenic tobacco plants, a novel chip-based capillary electrophoretic method (Experion), classical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) and a square wave voltammetric method using a carbon paste electrode. We determined that the automated chip-based capillary electrophoretic method is rapid, sensitive and the results obtained are well repeatable. The avidin content measured in transgenic tobacco leaves using chip-based capillary electrophoresis varied from 15 to 854 ng per mg of fresh mass depending on the individual plant.     

Fluorescence Dynamics of Monocyclodextrin– and Bis(thiol‐cyclodextrin)–Coumarin C153 Complexes

The solvation and confinement of coumarin C153 within supramolecular host/guest complexes based on β‐cyclodextrin (β‐CD) and 6‐deoxy‐6‐thio‐β‐cyclodextrin (β‐CD‐SH) in water are studied by fluorescence spectroscopy. For β‐CD/C153, the 1:1 complex is proposed, and for β‐CD‐SH/C153 both the 1:1 and 2:1 complexes are believed to be formed. The 2:1 β‐CD‐SH/C153 complex has an association constant of 4.2×10⁵ M ^?1 and a C153 population of 82 %, which are interestingly high values, indicating that the proposed β‐CD‐SH dimers structure are connected by covalent disulfide bonds; this is supported by mass spectrometry. Solvation related to fast hydrogen‐bond rearrangement as a part of fluorescence relaxation is determined by the ultrafast components of time‐resolved spectroscopy to be 3 and 7 ps for the 1:1 β‐CD/C153 and 2:1 β‐CD‐SH/C153 complexes, respectively.     

Comparison of Metallothionein Detection by Using Brdicka Reaction and Enzyme‐Linked Immunosorbent Assay Employing Chicken Yolk Antibodies

In this study, two methods for metallothioneins (MT) determination in a biological sample were compared. Particularly, twenty five human and nine pig blood serum samples and liver and kidney samples from thirty five carps (Cyprinus carpio) were analyzed by enzyme‐linked immunosorbent assay and differential pulse voltammetry Brdicka's reaction. The results obtained by these two methods were in good agreement. For commercially available MT standard the correlation coefficient between the single concentrations signal height was 0.99. In biological samples the correlation coefficients were 0.90 for fish liver and kidney samples, 0.91 for pig blood serum and 0.93 for human blood serum.     

Pd-Catalyzed Heterocyclization During Sonogashira Coupling: Synthesis of 3-Aryl-substituted Imidazo[2,1-b]thiazoles

Abstract

The reaction of 4,5-diphenyl-2-propargylmercaptoimidazole with various aryl iodides catalyzed by Pd-Cu in the presence of triethylamine as base in dimethyl formamide (DMF) leads to the formation of 3-aryl-substituted imidazo[2,1-b]thiazoles.     

TDPADg-factor measurement of the 21/2+ state in89zr

Theg-factor of the 21/2⁺ state in⁸⁹Zr has been determined to be 0.89(4). The TDPAD method has been applied in the reaction⁸⁵Rb(⁷Li, 3n)⁸⁹Zr at 28 MeV. The obtained value agrees with shell model predictions.