17,193 research outputs found

    Differential Cyclic Voltammetry - a Novel Technique for Selective and Simultaneous Detection using Redox Cycling Based Sensors

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    Redox cycling (RC) is an effect that is used to amplify electrochemical signals. However, traditional techniques such as cyclic voltammetry (CV) do not provide clear insight for a mixture of multiple redox couples while RC is applied. Thus, we have developed a new measurement technique which delivers electrochemical spectra of all reversible redox couples present based on concentrations and standard potentials. This technique has been named differential cyclic voltammetry (DCV). We have fabricated micrometer-sized interdigitated electrode (IDE) sensors to conduct DCV measurements in mixtures of 1mM catechol and 4mM [Ru(NH3)6]Cl3. To simulate the electrochemical behavior of these sensors we have also developed a finite element model (FEM) in Comsol®. The\ud experimental data corresponds to the calculated spectra obtained from simulations. Additionally, the measured spectra can be used to easily derive standard potentials and concentrations simultaneously and selectively.\u

    Oxidative Stress Detection With Escherichia Coli Harboring A katG\u27::lux Fusion

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    A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed. An E. coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke. An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG\u27::lux luminescence. Light emission was also induced by ethanol by an unexplained mechanism. A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide. It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards


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    Jan Eijkel is Associate Professor in microfluidics and nanofluidics in the BIOS/Lab on a Chip group (MESA+ Institute for Nanotechnology, University of Twente, The Netherlands). He studied pharmacy (University of Amsterdam) and theology (University of Utrecht) and obtained a Ph.D. in biosensor research at the University of Twente. His research interests include physical and colloid chemistry, electrochemistry, microseparation methods, and microplasma physics and applications. Geen abstract beschikbaa

    DNA sensing by electrocatalysis with hemoglobin

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    Electrocatalysis offers a means of electrochemical signal amplification, yet in DNA-based sensors, electrocatalysis has required high-density DNA films and strict assembly and passivation conditions. Here, we describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low-density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low-density DNA films. Electrocatalysis with methylene blue that is covalently tethered to the DNA by a flexible alkyl chain linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of a single cytosine-adenine (CA) mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: It is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling

    Microbial diversity in Baltic Sea sediments

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    This thesis focuses on microbial community structures and their functions in Baltic Sea sediments. First we investigated the distribution of archaea and bacteria in Baltic Sea sediments along a eutrophication gradient. Community profile analysis of 16S rRNA genes using terminal restriction length polymorphism (T-RFLP) indicated that archaeal and bacterial communities were spatially heterogeneous. By employing statistical ordination methods we observed that archaea and bacteria were structured and impacted differently by environmental parameters that were significantly linked to eutrophication. In a separate study, we analyzed bacterial communities at a different site in the Baltic Sea that was heavily contaminated with polyaromatic hydrocarbons (PAHs) and several other pollutants. Sediment samples were collected before and after remediation by dredging in two consecutive years. A polyphasic experimental approach was used to assess growing bacteria and degradation genes in the sediments. The bacterial communities were significantly different before and after dredging of the sediment. Several isolates collected from contaminated sediments showed an intrinsic capacity for degradation of phenanthrene (a PAH model compound). Quantititative real-time PCR was used to monitor the abundance of degradation genes in sediment microcosms spiked with phenanthrene. Although both xylE and phnAc genes increased in abundance in the microcosms, the isolates only carried phnAc genes. Isolates with closest 16S rRNA gene sequence matches to Exigobacterium oxidotolerans, a Pseudomonas sp. and a Gammaproteobacterium were identified by all approaches used as growing bacteria that are capable of phenanthrene degradation. These isolates were assigned species and strain designations as follows: Exiguobacterium oxidotolerans AE3, Pseudomonas fluorescens AE1 and Pseudomonas migulae AE2. We also identified and studied the distribution of actively growing bacteria along red-ox profiles in Baltic Sea sediments. Community structures were found to be significantly different at different red-ox depths. Also, according to multivariate statistical ordination analysis organic carbon, nitrogen, and red-ox potential were crucial parameters for structuring the bacterial communities on a vertical scale. Novel lineages of bacteria were obtained by sequencing 16S rRNA genes from different red-ox depths and sampling stations indicating that bacterial diversity in Baltic Sea sediments is largely unexplored

    An ultrasensitive photoelectrochemical nucleic acid biosensor

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    A simple and ultrasensitive procedure for non-labeling detection of nucleic acids is described in this study. It is based on the photoelectrochemical detection of target nucleic acids by forming a nucleic acid/photoreporter adduct layer on an ITO electrode. The target nucleic acids were hybridized with immobilized oligonucleotide capture probes on the ITO electrode. A subsequent binding of a photoreporter—a photoactive threading bis-intercalator consisting of two N,N′-bis(3-propyl-imidazole)-1,4,5,8-naphthalene diimides (PIND) linked by a [Formula: see text] (bpy = 2,2′-bipyridine) complex (PIND–Ru–PIND)—allowed for photoelectrochemical detection of the target nucleic acids. The extremely low dissociation rate of the adduct and the highly reversible photoelectrochemical response under visible light illumination (490 nm) make it possible to conduct nucleic acid detection, with a sensitivity enhancement of four orders of magnitude over voltammetry. These results demonstrate for the first time the potential of photoelectrochemical biosensors for PCR-free ultrasensitive detection of nucleic acids

    Nanoscale Electrodes for Bionanosensing

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    Cancer is globally the second most common cause of death. Cancer burden rises to about 10 million deaths and more than 18 million new cases in 2018. Cancers are often diagnosed at a later stage preventing curative treatment. This underscores the need for an early stage diagnosis of cancer. Consequently, screening methods that can test patients’ samples taken by less invasive methods capable of early stage diagnosis are highly sought for. Based on this motivation, here we developed lab-on-a-chip diagnostic systems that can be used for early detection of cancer. Three different types of nanoscale electrodes were fabricated: (i) nanogap electrodes (ii) nano interdigitated electrodes and (iii) nanodisc electrodes and the possibility of using them for sensing and signal transduction were investigated. Chapter 2 describes the fabrication of nanogap device using conventional optical lithography and DNA detection across it using the electrical method. Chapter 3 details the fabrication of nano interdigitated electrodes (nIDEs) and their electrochemical validation. Chapter 4 describes the biosensing application of nIDEs using nanoparticle sandwich assay for the detection of DNA molecules. Chapter 5 describes the capturing of tdEVs on nIDEs, and its quantification using a sandwich immunosorbent assay on nIDEs. Chapter 6 proposes a new type of nanoscale electrodes which are termed as nanodisc electrodes. Chapter 7 explores the possibility of developing the nanodisc technology to a business idea. In short, the whole thesis tries to explore the different possibilities in developing a sensor that can be useful for cancer diagnosis

    Increased variability of microbial communities in restored salt marshes nearly 30 years after tidal flow restoration

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    We analyzed microbial diversity and community composition from four salt marsh sites that were impounded for 40–50 years and subsequently restored and four unimpounded sites in southeastern Connecticut over one growing season. Community composition and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) and sequence analysis of 16S ribosomal RNA (rRNA) genes. Our results indicated diverse communities, with sequences representing 14 different bacterial divisions. Proteobacteria, Bacteroidetes, and Planctomycetes dominated clone libraries from both restored and unimpounded sites. Multivariate analysis of the TRFLP data suggest significant site, sample date, and restoration status effects, but the exact causes of these effects are not clear. Composition of clone libraries and abundance of bacterial 16S rRNA genes were not significantly different between restored sites and unimpounded sites, but restored sites showed greater temporal and spatial variability of bacterial communities based on TRFLP profiles compared with unimpounded sites, and variability was greatest at sites more recently restored. In summary, our study suggests there may be long-lasting effects on stability of bacterial communities in restored salt marshes and raises questions about the resilience and ultimate recovery of the communities after chronic disturbance

    Similar Microbial Communities Found on Two Distant Seafloor Basalts.

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    The oceanic crust forms two thirds of the Earth's surface and hosts a large phylogenetic and functional diversity of microorganisms. While advances have been made in the sedimentary realm, our understanding of the igneous rock portion as a microbial habitat has remained limited. We present the first comparative metagenomic microbial community analysis from ocean floor basalt environments at the Lō'ihi Seamount, Hawai'i, and the East Pacific Rise (EPR; 9°N). Phylogenetic analysis indicates the presence of a total of 43 bacterial and archaeal mono-phyletic groups, dominated by Alpha- and Gammaproteobacteria, as well as Thaumarchaeota. Functional gene analysis suggests that these Thaumarchaeota play an important role in ammonium oxidation on seafloor basalts. In addition to ammonium oxidation, the seafloor basalt habitat reveals a wide spectrum of other metabolic potentials, including CO2 fixation, denitrification, dissimilatory sulfate reduction, and sulfur oxidation. Basalt communities from Lō'ihi and the EPR show considerable metabolic and phylogenetic overlap down to the genus level despite geographic distance and slightly different seafloor basalt mineralogy

    The compositional and evolutionary logic of metabolism

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    Metabolism displays striking and robust regularities in the forms of modularity and hierarchy, whose composition may be compactly described. This renders metabolic architecture comprehensible as a system, and suggests the order in which layers of that system emerged. Metabolism also serves as the foundation in other hierarchies, at least up to cellular integration including bioenergetics and molecular replication, and trophic ecology. The recapitulation of patterns first seen in metabolism, in these higher levels, suggests metabolism as a source of causation or constraint on many forms of organization in the biosphere. We identify as modules widely reused subsets of chemicals, reactions, or functions, each with a conserved internal structure. At the small molecule substrate level, module boundaries are generally associated with the most complex reaction mechanisms and the most conserved enzymes. Cofactors form a structurally and functionally distinctive control layer over the small-molecule substrate. Complex cofactors are often used at module boundaries of the substrate level, while simpler ones participate in widely used reactions. Cofactor functions thus act as "keys" that incorporate classes of organic reactions within biochemistry. The same modules that organize the compositional diversity of metabolism are argued to have governed long-term evolution. Early evolution of core metabolism, especially carbon-fixation, appears to have required few innovations among a small number of conserved modules, to produce adaptations to simple biogeochemical changes of environment. We demonstrate these features of metabolism at several levels of hierarchy, beginning with the small-molecule substrate and network architecture, continuing with cofactors and key conserved reactions, and culminating in the aggregation of multiple diverse physical and biochemical processes in cells.Comment: 56 pages, 28 figure