dna-magnetic-particle-binding-analysis-dynamic-electrophoretic-light

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Mendel University in Brno

Brno University of Technology

Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of -0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process.

The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.

The human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) are carriers of neurotransmitters and targets for many drugs. Pioneering works in the past three years to elucidate experimental models of the Drosophila dDAT and human hSERT structures will rapidly impact the field of neuroscience. Here, we evaluated automated homology-based human models of these transporters, employing systematic physics-based, knowledge-based, and empirical-based check. Modeling guidelines were conveyed with attention to the central binding site (S1), secondary binding site (S2), and the extracellular loops EL2 and EL4. Application of new experimental models (dDAT and hSERT) will improve the accuracy of homology models, previously utilizing prokaryotic leucine transporter (LeuT) structure, and provide better predictions of ligand interactions, which is required for understanding of cellular mechanisms and for development of novel therapeutics.

Guidelines for Homology Modeling of Dopamine, Norepinephrine, and Serotonin Transporters.

Prostate cancer cells control energy metabolism by chelating intracellular zinc. Thus, zinc delivery has been a popular therapeutic approach for prostate cancer. Here, we propose the use of the membrane-penetrating peptide Novicidin connected to zinc-Schiff base as a carrier vehicle for the delivery of zinc to prostate cells. Mass spectrometry, electrochemistry and spectrophotometry confirmed the formation/stability of this complex and provided insight regarding the availability of zinc for complex interactions. This delivery system showed minor toxicity in normal PNT1A cells and high potency towards PC3 tumor cells. The complex preferentially penetrated PC3 tumor cells in contrast to confinement to the membranes of PNT1A. Furthermore, zinc uptake was confirmed in both cell lines. Molecular analysis was used to confirm the activation of zinc stress (e.g., ZnT-1) and apoptosis (e.g., CASP-1). Our results strongly suggest that the zinc-Schiff base-Novicidin complex has great potential as a novel anticancer drug.

The Zinc-Schiff Base-Novicidin Complex as a Potential Prostate Cancer Therapy.

Herein we describe a novel alternative synthesis route of polyvinylpyrrolidone nanoparticles using salting-out method at a temperature close to polyvinylpyrrolidone decomposition. At elevated temperatures, the stability of polyvinylpyrrolidone decreases and the opening of pyrrolidone ring fractions occurs. This leads to cross-linking process, where separate units of polyvinylpyrrolidone interact among themselves and rearrange to form nanoparticles. The formation/stability of these nanoparticles was confirmed by transmission electron microscopy, X-ray photoelectron spectroscopy, mass spectrometry, infrared spectroscopy, and spectrophotometry. The obtained nanoparticles possess exceptional biocompatibility. No toxicity and genotoxicity was found in normal human prostate epithelium cells (PNT1A) together with their high hemocompatibility. The antimicrobial effects of polyvinylpyrrolidone nanoparticles were tested on bacterial strains isolated from the wounds of patients suffering from hard-to-heal infections. Molecular analysis (qPCR) confirmed that the treatment can induce the regulation of stress-related survival genes. Our results strongly suggest that the polyvinylpyrrolidone nanoparticles have great potential to be developed into a novel antibacterial compound.

Alternative Synthesis Route of Biocompatible Polyvinylpyrrolidone Nanoparticles and Their Effect on Pathogenic Microorganisms.

In the last decade, the control of avian influenza virus has experienced many difficulties, which have caused major global agricultural problems that have also led to public health consequences. Conventional biochemical methods are not sufficient to detect and control agricultural pathogens in the field due to the growing demand for food and subsidiary products; thus, studies aiming to develop potent alternatives to conventional biochemical methods are urgently needed. In this review, emerging detection systems, their applicability to diagnostics, and their therapeutic possibilities in view of nanotechnology are discussed. Nanotechnology-based sensors are used for rapid, sensitive and cost-effective diagnostics of agricultural pathogens. The application of different nanomaterials promotes interactions between these materials and the virus, which enables researchers to construct portable electroanalytical biosensing analyser that should effectively detect the influenza virus. The present review will provide insights into the guidelines for future experiments to develop better techniques to detect and control influenza viruses.

Advanced nanotechnologies in avian influenza: Current status and future trends - A review.

The increasing number of scientific publications focusing on magnetic materials indicates growing interest in the broader scientific community. Substantial progress was made in the synthesis of magnetic materials of desired size, morphology, chemical composition, and surface chemistry. Physical and chemical stability of magnetic materials is acquired by the coating. Moreover, surface layers of polymers, silica, biomolecules, etc. can be designed to obtain affinity to target molecules. The combination of the ability to respond to the external magnetic field and the rich possibilities of coatings makes magnetic materials universal tool for magnetic separations of small molecules, biomolecules and cells. In the biomedical field, magnetic particles and magnetic composites are utilized as the drug carriers, as contrast agents for magnetic resonance imaging (MRI), and in magnetic hyperthermia. However, the multifunctional magnetic particles enabling the diagnosis and therapy at the same time are emerging. The presented review article summarizes the findings regarding the design and synthesis of magnetic materials focused on biomedical applications. We highlight the utilization of magnetic materials in separation/preconcentration of various molecules and cells, and their use in diagnosis and therapy.

Yazan Haddad

Department of Chemistry and Biochemistry

Mendel University in Brno

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering