Electrically Guided DNA Immobilization and Multiplexed DNA Detection with Nanoporous Gold Electrodes.

Molecular diagnostics have significantly advanced the early detection of diseases, where the electrochemical sensing of biomarkers (e.g., DNA, RNA, proteins) using multiple electrode arrays (MEAs) has shown considerable promise. Nanostructuring the electrode surface results in higher surface coverage of capture probes and more favorable orientation, as well as transport phenomena unique to nanoscale, ultimately leading to enhanced sensor performance. The central goal of this study is to investigate the influence of electrode nanostructure on electrically-guided immobilization of DNA probes for nucleic acid detection in a multiplexed format. To that end, we used nanoporous gold (np-Au) electrodes that reduced the limit of detection (LOD) for DNA targets by two orders of magnitude compared to their planar counterparts, where the LOD was further improved by an additional order of magnitude after reducing the electrode diameter. The reduced electrode diameter also made it possible to create a np-Au MEA encapsulated in a microfluidic channel. The electro-grafting reduced the necessary incubation time to immobilize DNA probes into the porous electrodes down to 10 min (25-fold reduction compared to passive immobilization) and allowed for grafting a different DNA probe sequence onto each electrode in the array. The resulting platform was successfully used for the multiplexed detection of three different biomarker genes relevant to breast cancer diagnosis

Anomalous Trends in Nucleic Acid-Based Electrochemical Biosensors with Nanoporous Gold Electrodes

Molecular diagnostics have significantly advanced the early detection of diseases, where electrochemical sensing of biomarkers has shown considerable promise. For a nucleic acid-based electrochemical sensor with signal-off behavior, the performance is evaluated by percent signal suppression (% ss), which indicates the change in current after hybridization. The % ss is generally due to more redox molecules (e.g., methylene blue) associating with the probe DNA bases in the single-strand form than the double-strand form upon hybridization with the target nucleic acid. Nanostructured electrodes generally enhance electrochemical sensor performance via several mechanisms, including increased number of capture probes per electrode volume and unique nanoscale transport phenomena. Here, we employ nanoporous gold (np-Au) as a model electrode material to study the influence of probe immobilization solution concentration on sensor performance and the underlying mechanisms. Unlike planar gold (pl-Au) electrodes, where % ss reaches a steady state with increasing concentration of the grafting solution, the % ss displays peak performance at certain grafting solution concentrations followed by rapid deterioration and reversal of the % ss polarity, suggesting an unexpected case of increased charge transfer upon hybridization. Fluorometric assessments of electrochemically desorbed nucleic acids for different electrode morphologies reveal that a significant amount of DNA molecules (unhybridized and hybridized) remain within the nanopores posthybridization. Analysis of electrochemical signals (e.g., square wave voltammogram shape) suggests that the large unbound nucleic acid concentration may be altering the modes of methylene blue interaction with the nucleic acids and charge transfer to the electrode surfaces

Interplay of Effective Surface Area, Mass Transport, and Electrochemical Features in Nanoporous Nucleic Acid Sensors

Electrochemical biosensors transduce biochemical events (e.g., DNA hybridization) to electrical signals and can be readily interfaced with electronic instrumentation for portability. Nanostructuring the working electrode enhances sensor performance via augmented effective surface area that increases the capture probability of an analyte. However, increasing the effective surface area via thicker nanostructured electrodes hinders the analyte's permeation into the nanostructured volume and limits its access to deeper electrode surfaces. Here, we use nanoporous gold (np-Au) with various thicknesses and pore morphologies coupled with a methylene blue (MB) reporter-tagged DNA probe for DNA target detection as a model system to study the influence of electrode features on electrochemical sensing performance. Independent of the DNA target concentration, the hybridization current (surrogate for detection sensitivity) increases with the surface enhancement factor (EF), until an EF of ∼5, after which the sensor performance deteriorates. Electrochemical and fluorometric quantification of a desorbed DNA probe suggest that DNA permeation is severely limited for higher EFs. In addition, undesirable capacitive currents disguise the faradaic currents from the MB reporter at larger EFs that require higher square wave voltammetry (SWV) frequencies. Finally, a real-time hybridization study reveals that expanding the effective surface area beyond EFs of ∼5 decreases sensor performance

Bactericidal and virucidal ultrathin films assembled layer by layer from polycationic N-alkylated polyethylenimines and polyanions

In this work, we designed contact-killing ionically cross-linked polymeric thin films using Layer-by-Layer (LbL) technology. A polycation, N,N-dodecyl,methyl-polyethylenimine, with microbicidal activity was layered with a polyanion, such as poly(acrylic acid), to create LbL films highly effective against both airborne and waterborne Escherichia coli and Staphylococcus aureus (Gram negative and positive bacteria, respectively), as well as influenza A/WSN (H1N1) virus. The dependence of the microbicidal activity on the pH during and post-assembly of LbL film formation, the nature of the polycation and polyanion, the number of layers in the LbL film, and other experimental variables was investigated quantitatively.close343

Assessment of Life Quality in Children with Dysphonia Using Modified Pediatric Voice-Related Quality of Life Questionnaire in Serbia

(1) Background: Hoarseness is not uncommon in children, especially at school age, as communication with peers is intensified. It is caused by improper use or overuse of the vocal apparatus. (2) Methods: The study included 85 hoarse children aged 6–12 (study group) and 240 healthy children (control group) of the same age. The study group underwent a detailed medical history, phoniatric examination, larynx fiber endoscopy, allergy treatment and the Pediatric Voice-Related Quality of Life questionnaire, modified by Jasmina Stojanovic. (3) Results: Our modified questionnaire revealed the significance of parental perception of a voice disorder in a child after organized activities. Using our modified questionnaire, we were able to determine the most frequent form of a voice disorder in children—speaking too loudly—is often neglected by the environment and can lead to an overall lower life quality. (4) Conclusions: As the presence of hoarseness impairs the quality of life in the pediatric population, awareness of a voice disorder must be recognized and treated on time to overcome the possible side effects on a child’s psychological and emotional development

The Monte Carlo Method Based on Eclectic Data as an Efficient Tool for Predictions of Endpoints for Nanomaterials - Two Examples of Application

The theoretical predictions of endpoints related to nanomaterials are attractive and moreefficient alternatives for their experimental determinations. Such type of calculations for the "usual"substances (i.e. non nanomaterials) can be carried out with molecular graphs. However, in the case ofnanomaterials, descriptors traditionally used for the quantitative structure - property/activityrelationships (QSPRs/QSARs) do not provide reliable results since the molecular structure ofnanomaterials, as a rule, cannot be expressed by the molecular graph. Innovative principles ofcomputational prediction of endpoints related to nanomaterials extracted from available eclectic data(technological attributes, conditions of the synthesis, etc.) are suggested, applied to two different sets of data, anddiscussed in this work.Fil: Toropov, Andrei A.. IRCCS. Istituto di Ricerche Farmacologiche Mario Negri; ItaliaFil: Toropova, Alla P.. IRCCS. Istituto di Ricerche Farmacologiche Mario Negri; ItaliaFil: Veselinovic, Alexander M.. University of Nis; SerbiaFil: Veselinovic, Jovana B.. University of Nis; SerbiaFil: Nesmerak, Karel. Charles University in Prague. Faculty of Science; República ChecaFil: Raska, Ivan, J.. Charles University in Prague. 3rd Department of Medicine; República ChecaFil: Duchowicz, Pablo Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina. Universidad Nacional de la Plata; ArgentinaFil: Castro, Eduardo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina. Universidad Nacional de la Plata; ArgentinaFil: Kudyshkin, Valentin O.. Uzbek Academy of Sciences; UzbekistánFil: Lleszczynska, Danuta. Jackson State University; Estados UnidosFil: Leszczynski, Jerzy. Jackson State University; Estados Unido

Multilayer Films Assembled from Naturally-Derived Materials for Controlled Protein Release

Herein we designed and characterized films composed of naturally derived materials for controlled release of proteins. Traditional drug delivery strategies rely on synthetic or semisynthetic materials or utilize potentially denaturing assembly conditions that are not optimal for sensitive biologics. Layer-by-layer (LbL) assembly of films uses benign conditions and can generate films with various release mechanisms including hydrolysis-facilitated degradation. These use components such as synthetic polycations that degrade into non-natural products. Herein we report the use of a naturally derived, biocompatible and degradable polyanion, poly(β-l-malic acid), alone and in combination with chitosan in an LbL film, whose degradation products of malic acid and chitosan are both generally recognized as safe (GRAS) by the FDA. We have found that films based on this polyanion have shown sustained release of a model protein, lysozyme that can be timed from tens of minutes to multiple days through different film architectures. We also report the incorporation and release of a clinically used biologic, basic fibroblast growth factor (bFGF), which demonstrates the use of this strategy as a platform for controlled release of various biologics.United States. Army Research Office (Contract W911NF-13-D-0001)United States. Air Force (Contract W911NF-07-D-0004)National Institutes of Health (U.S.) (National Cancer Institute (U.S.) U01 CA151815

Biological activity of Pinus nigra terpenes-Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity

In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50=25.08 mg/mL and VitC=0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity. (C) 2014 Elsevier Ltd. All rights reserved.Ministry of Education and Science of the Republic of Serbia {[}173029