Cholinesterase based amperometric biosensors for assay of anticholinergic compounds

Biosensors are analytical devices being approachable for multiple analytes assay. Here, biosensors with intercepted acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) are presented as tool for assay of anticholinergic compounds such as pesticides, nerve agents and some natural toxins. Principle of assay is based on evaluation of cholinesterase activity and its pertinent decrease in presence of analyte. Nerve agents, pesticides, anticholinergic drugs useable for treatment of Alzheimer′s disease as well as myasthenia gravis and aflatoxins are enlisted as compounds simply analyzable by cholinesterase biosensors

Nephele: genotyping via complete composition vectors and MapReduce

<p>Abstract</p> <p>Background</p> <p>Current sequencing technology makes it practical to sequence many samples of a given organism, raising new challenges for the processing and interpretation of large genomics data sets with associated metadata. Traditional computational phylogenetic methods are ideal for studying the evolution of gene/protein families and using those to infer the evolution of an organism, but are less than ideal for the study of the whole organism mainly due to the presence of insertions/deletions/rearrangements. These methods provide the researcher with the ability to group a set of samples into distinct genotypic groups based on sequence similarity, which can then be associated with metadata, such as host information, pathogenicity, and time or location of occurrence. Genotyping is critical to understanding, at a genomic level, the origin and spread of infectious diseases. Increasingly, genotyping is coming into use for disease surveillance activities, as well as for microbial forensics. The classic genotyping approach has been based on phylogenetic analysis, starting with a multiple sequence alignment. Genotypes are then established by expert examination of phylogenetic trees. However, these traditional single-processor methods are suboptimal for rapidly growing sequence datasets being generated by next-generation DNA sequencing machines, because they increase in computational complexity quickly with the number of sequences.</p> <p>Results</p> <p>Nephele is a suite of tools that uses the complete composition vector algorithm to represent each sequence in the dataset as a vector derived from its constituent k-mers by passing the need for multiple sequence alignment, and affinity propagation clustering to group the sequences into genotypes based on a distance measure over the vectors. Our methods produce results that correlate well with expert-defined clades or genotypes, at a fraction of the computational cost of traditional phylogenetic methods run on traditional hardware. Nephele can use the open-source Hadoop implementation of MapReduce to parallelize execution using multiple compute nodes. We were able to generate a neighbour-joined tree of over 10,000 16S samples in less than 2 hours.</p> <p>Conclusions</p> <p>We conclude that using Nephele can substantially decrease the processing time required for generating genotype trees of tens to hundreds of organisms at genome scale sequence coverage.</p

Enzymatic Glucose Based Bio batteries: Bioenergy to Fuel Next Generation Devices

[EN] This article consists of a review of the main concepts and paradigms established in the field of biological fuel cells or biofuel cells. The aim is to provide an overview of the current panorama, basic concepts, and methodologies used in the field of enzymatic biofuel cells, as well as the applications of these bio-systems in flexible electronics and implantable or portable devices. Finally, the challenges needing to be addressed in the development of biofuel cells capable of supplying power to small size devices with applications in areas related to health and well-being or next-generation portable devices are analyzed. The aim of this study is to contribute to biofuel cell technology development; this is a multidisciplinary topic about which review articles related to different scientific areas, from Materials Science to technology applications, can be found. 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A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

Abstract PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications

A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications

Calibration curves generated with either the purified 196<i>mecA</i> amplicon (diamonds) or the unpurified PCR mixture (containing the target amplicon) (squares).

<p>The logarithmic trendlines were calculated in Excel, and proved to be identical for the purified and non-purified amplicons.</p

The oligonucleotide probe and targets used in the current study.

<p>Capital letters show sequences that are cognate between a target oligonucleotide and the probe, with the restriction site shown in bold.</p>1<p>The total length of a target sequence that is complementary to the 40-mer probe MCA-BG.</p>2<p>Tm was calculated for a target-probe hybrid in PBS (150 mM Na⁺).</p

Detection of the non-purified amplicon <i>mecA</i> in the presence of a large excess of heterologous (mouse) genomic DNA.

<p>Circles and diamonds show replicate experiments performed using the amplicon-containing PCR mixture, closed and open for addition of 100 or 0 ng of mouse DNA, respectively. The triangles show the negative control supplemented with 100 ng of mouse DNA, specifically, dilutions of the whole PCR mixture that were not subjected to thermocycling (no amplicon formation as verified by gel electrophoresis).</p

Effect of point mutations introduced into the target sequence.

<p>(<b>A</b>) Single, double and triple mutations were introduced between the target center and the 3′ end corresponding to the surface-immobilized terminus of the target-probe duplex. (<b>B</b>) Mutations were introduced between the target center and the 5′ end corresponding to the end of the target-probe duplex that was free in solution. HRP signals (bars) are expressed as the percentages of the fully cognate positive control (dark grey bar 40, for 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a 40-mer target with 1–3 mutations shown with black ovals. The <i>Bgl</i>II restriction site is indicated with thick horizontal lines. Targets are named with ‘rs’ for mutations introduced within the restriction site, otherwise the target name contains the replacement nucleotide (mostly G) and position within the sequence, starting from the 5′ target end. The rs19+24 contained two mutations at the ends of the restriction site. Target oligonucleotide sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097826#pone-0097826-t001" target="_blank">Table 1</a>.</p