Preparation and optimization of a bienzymic biosensor based on self-assembled monolayer modified gold electrode for alcohol and glucose detection

PubMed ID: 19819124The aim of this project was to develop a bienzymic biosensor, which was based on co-immobilization of alcohol oxidase and glucose oxidase on the same electrode by formation of self-assembled monolayer (SAM) for selective determination of ethanol and glucose. In the biosensor construction the enzymes and the mediator, tetrathiafulvalene (TTF), were immobilized with cross-linking agents glutaraldehyde and cysteamine by forming a self-assembled monolayer (SAM) on a gold disc electrode. Amounts of ethanol and glucose were amperometrically detected by monitoring current values at reduction potential of TTF+, 0.1 V. Decreases in biosensor responses were linearly related to glucose concentrations between 0.1 and 1.0 mM and ethanol concentrations between 1.0 and 10 mM. Limits of detection of the biosensor for ethanol and glucose were calculated to be 0.75 and 0.03 mM, respectively. In the optimization studies of the biosensor some parameters such as optimum pH, optimum temperature, enzyme amount, effect of TTF concentration and duration of SAM formation were investigated. © 2009 Elsevier B.V. All rights reserved.2008 FEN 014This project was funded by Ege University Research Fund (project no.: 2008 FEN 014). -

Tempo and Mode of Gene Duplication in Mammalian Ribosomal Protein Evolution

<div><p>Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism.</p></div

Selective Pressures on Ribosomal Protein Gene Duplicates.

<p>A) Mean Ka/Ks ratios were calculated for all classes (DD-RPs, RT-RPs and RΨ-RPs) of RP gene duplicates using the Nei Gojobori method. Results were then filtered based on p-values (<0.1) and the fraction of the parental gene represented by each duplicate (>65%). Error bars represent 95% confidence interval. B) Box and whisker plots for RT-RPs (blue) and RΨ-RPs (green) were generated for inner speciation nodes and C) Extant Species. DD-RPs were not included in the analyses due to small sample size (N = 3).</p

Example of the inferred evolutionary history for duplications of the ribosomal protein gene Rpl36a.

<p>Grey outlined tube tree represents the species tree that includes 8 mammals and chicken. Parental intron-bearing gene (in blue). RT-RPs (clear triangles), RΨ-RPs (grey triangles). An RT-RP duplicate generated from one of these events, Rpl36al (in red, at the base of the mammalian lineage on the branch between LCA with opossum and the other mammals) is conserved in all descendent species. All the 74 ribosomal protein gene family history trees are attached as supplementary material.</p

Pipeline for ribosomal protein family analyses.

<p>Protein sequence for all parental ribosomal proteins were collected manually from Ensembl62. These were input to tBLASTn against whole genomes to capture all putative duplicates. The resulting duplicates were processed by Pseudopipe to determine the mechanism of duplication (DNA or RNA) and the fate of the duplicate (intact or pseudogenized). We then utilized our in-house pipeline steps of hierarchical clustering by local synteny³ in order to build our gene family trees after filtering false-positives and redundant entries. Final gene family analyses were conducted in 2 steps: 1) calculating the selective pressures on all gene duplicates using the Nei-Gojobori method against the species- and family-specific seed protein via an exon-based reconstruction, and 2) checking for expression signatures via EST analyses using the UCSC genome browser EST track for both human and mouse.</p

Ribosomal Protein Family duplication events based on age.

<p>All RP gene duplication events are displayed for 8 mammalian species. The bar charts at all speciation nodes show events classified by fate of duplication. The duplication counts on the bar charts are log normalized. RT-RPs are shown in red and RΨ-RPs in green. DD-RPs are not shown due to a very small sample size. The numbers above the bar charts represent the total number of gene duplication events at that speciation node. Age is marked at the bottom of the tree in millions of years (age estimates from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111721#pone.0111721-Mikkelsen1" target="_blank">[55]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111721#pone.0111721-UretaVidal2" target="_blank">[103]</a>).</p

RP gene duplicates in 8 mammalian genomes.

<p>A) Distribution of duplication events in 8 mammalian genomes. B) Assessment of coverage or species-specific bias in ribosomal protein gene duplicates. C) Representation of DNA and RNA-mediated duplications in RP gene families. Abbreviations: Hs, <i>Homo sapiens</i> (human); Pt, <i>Pan troglodytes</i> (chimpanzee); Mmul, <i>Macaca mulatta</i> (Rhesus macaque); Mm, <i>Mus musculus</i> (house mouse); Rn, <i>Rattus norvegicus</i> (Norway rat); Bt, <i>Bos taurus</i> (cattle); Cf, <i>Canis familiaris</i> (dog); Md, <i>Monodelphis domestica</i> (gray short-tailed opossum); Gg, <i>gallus gallus</i> (chicken).</p

Likelihood Ratio Test statistics (LRT) for random site models.

a<p>Alternative model;</p>b<p>null model; 2Δℓ = 2(ℓ₁−ℓ₀), df degrees of freedom.</p><p>Likelihood Ratio Test statistics (LRT) for random site models.</p