One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method

[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.This research has been funded through Projects GVA-PROMETEO/2010/008 (Generalitat Valenciana) and CTQ/2013/ 45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2014). One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method. Sensors and Actuators B: Chemical. 204:273-281. https://doi.org/10.1016/j.snb.2014.07.073S27328120

Atomic force microscopy based nanoassay: A new method to study \u3b1-Synuclein-dopamine bioaffinity interactions

Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular \u3b1-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of \u3b1-Synuclein on gold to study the early stages of \u3b1-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-\u3b1-Synuclein adducts

Thermal Performance of Tropical Atrium

Atrium is a popular architectural feature utilized widely by building designers and owners to bring various benefits such as adequate daylight, circulation spaces and surfaces for landscape applications. But atrium problems in tropical climates such as excessive daylight, glare and high temperature, which lead to increase building energy demand, have been reported. To avoid and reduce these unpleasant features, a side-lit atrium has been suggested. Although researchers proposed side-lit atrium to prevent common problems of atria, the lack of precedent research on this issue compels these authors to study atrium performance in hot and humid climate. So the research aims to examine two different atrium roof form types in terms of temperature and ventilation impacts in hot and humid climate of Malaysia using DesignBuilder as a simulation program. The results indicate lower temperature of side-lit model with better airflow pattern in comparison with top-lit model while the top-lit model provides higher air velocity at the air inlet and outlet

Surface–Enhanced Raman Spectroscopy Label–free Detection of DNA Complementary PolyA and PolyT Polybases on Ag/TiO2 Platform

International audienceAn original, robust, and low cost Ag°/TiO2 platform has been used for the Surface-Enhanced Raman Spectroscopy (SERS) detection of polyA-NH2 and polyT complementary bases. Three procedures have been assessed to immobilize the polybases in various concentrations on the SERS platform, namely immobilization of polybases alone, immobilization of the polyA-NH2+polyT mixture, and two-step immobilization of the polyA-NH2•polyT system. Derived SERS spectra have been analyzed and are discussed in terms of spectrum reproducibility, influence of polybase concentration in solution, orientation of polybases at the SERS platform surface, as well as feasibility of complementary polybase hybridization. This study allows us envisaging a simple hybridization procedure using the polyA-NH2•polyT system and an efficient SERS detection of in situ hybridization features through intensity measurements of the adenine vibration mode at 1330 cm−1