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

Forest carbon sequestration:the impact of forest management

In this chapter, we describe alternative ways in which forests and forestry can help to mítigate climate change, along with the potential impact of these activities. The three carbon storage compartments should be considered inall impact estimates. Carbon content in living biomass is easily estimated via species-specific equations or by applying factors to oven-dry biomass weights (e.g.,lbañez et al.,2002, Herrero et al.,2011,Castaño and Bravo, 2012).Litter carbon content has been analysed in many studies on primary forest productivity, though information regarding the influence of forest management on litter carbon content is less abundant (Blanco et al., 2006). In the last decade,efforts have been made to assess soil carbon in forests, but studies on the effect of forest management on soils show discrepancies (Lindner and Karjalainen,2007).Hoover (2011), for example,found no difference in forest floor carbon stocks among stands subjected to partial or complete harvest treatments in the United States.Instituto Universitario de Gestión Forestal Sostenibl

DNA biosensors based on integrated isothermal amplification-detection strategies

L'objectiu principal d'aquesta Tesi Doctoral és presentar un enfoc diferent i alternatiu dels sensors d'ADN per dissenyar i fabricar un nou tipus de plataforma que combini l'amplificació amb la detecció d'ADN, alhora que es superen algunes de les limitacions existents del camp. Per aconseguir aquest objectiu, es presenten una sèrie d'estratègies basades en explotar noves vies d'immobilització en fase sòlida i amplificació enzimàtica per aconseguir millors límits de detecció mitjançant assajos ràpids i fàcils d'executar. El treball exposat representa un mètode ràpid, simple i fàcil d'integrar alhora que és una plataforma de biodetecció que aporta noves idees per a la integració dels assajos d'ADN en dispositius d'intervenció en el llocs d'interès a través de solucions miniaturitzades o "lab on a xip ". La visió que es persegueix en última instància al llarg d'aquest treball és la humil contribució en aquest concepte lligat a la descentralització, permetent que el camp del diagnòstic molecular es mogui fora del laboratori per a proveir informació bioanalítica in situ. La Tesi descriu l'ús d'estratègies d'amplificació amb polimerases i recombinasas en fases sòlides com a concepte híbrid que combina l'amplificació i la detecció d'ADN, la detecció de mostres reals, l'explotació dels límits de la química de superfície i de l'ADN per desenvolupar estratègies que supleixin les limitacions trobades i l'ús de "ring resonators" per monitoritzar l'evolució d'aquest mecanisme sense marcadors i en temps real.El objetivo principal de esta Tesis Doctoral es presentar un distinto enfoque y alternativa de los sensores de ADN para diseñar y fabricar un nuevo tipo de plataforma que combine la amplificación con la detección de ADN, a la vez que se superan algunas de las limitaciones existentes en el campo. Para conseguir dicho objetivo, se presentan una serie de estrategias distintas basadas en explotar nuevas estrategias de inmovilización en fase sólida y amplificación enzimática para conseguir mejores límites de detección mediante ensayos rápidos y fáciles de ejecutar. El trabajo expuesto representa un método rápido, simple y fácil de integrar a la vez que es una plataforma de biodetección que aporta nuevas ideas para la integración de los ensayos de ADN en dispositivos intervención en el sitio de interés a través de soluciones miniaturizadas o “lab on a chip”. La visión que se persigue en última instancia a lo largo de este trabajo es la humilde contribución en este concepto ligado a la descentralización, permitiendo que el campo de diagnóstico molecular se mueva fuera del laboratorio para proveer información bioanalítica in situ. La Tesis describe el uso de estrategias de amplificación con polimerasas y recombinasas en fases sólidas como concepto híbrido que combina la amplificación y la detección de ADN, la detección de muestras reales, la explotación de los límites de la química de superficie y del ADN para desarrollar estrategias que suplan las limitaciones encontradas y el uso de “ring resonators” para monitorizar la evolución de dicho mecanismo sin marcadores y en tiempo real.The main goal of this Doctoral Thesis is to present alternative approaches in the field of DNA biosensors, designing and building new detection platforms that combine the amplification and quantification of targeted DNA while overcoming some of the current limitations. In order to achieve this objective, a variety of different strategies of effective solid-phase immobilisation strategies and isothermal enzymatic amplification have been explored to achieve lower detection limits with rapid and easy to execute assays. This work presents a convenient, rapid, simple, easy to integrate yet robust biosensing detection platform that can bring new ideas for the integration of nucleic acid tests in point of care devices through the use of lab on a chip solutions. Ultimately, the vision underlining this work is to humbly contribute to the concept of decentralisation, allowing molecular diagnostics to move away from laboratories, providing bioanalytical information in situ. The Thesis report the work performed to achieve the specific objectives of this Doctoral Thesis: the use of solid-phase recombinase polymerase amplification strategy, as a hybrid concept that combines DNA amplification and detection, the detection of real samples, the exploiting of the surface chemistry and DNA to overcome the limitations found, and the use of ring resonators for the label-free and real-time monitoring of the RPA mechanism

An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids

© 2019, The Author(s), under exclusive licence to Springer Nature Limited.Affinity-based electrochemical detection in complex biological fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 month of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy preparation, stability and simplicity of this nanocomposite allow the generation of electrochemical biosensors that can operate in complex biological fluids such as blood plasma or serum11sciescopu

Organoid‐Based Human Stomach Micro‐Physiological System to Recapitulate the Dynamic Mucosal Defense Mechanism

Abstract Several stomach diseases are attributed to the dysregulation of physiological function of gastric mucosal barrier by pathogens. Gastric organoids are a promising tool to develop treatment strategies for gastric infections. However, their functional features of in vivo gastric mucosal barrier and host–microbe interactions are limited due to the lack of physiological stimuli. Herein, a human stomach micro‐physiological system (hsMPS) with physiologically relevant gastric mucosal defense system is described based on the combination of organoid and MPS technology. A fluid flow enhanced epithelial‐mesenchymal interaction in the hsMPS enables functional maturation of gastric epithelial cells, which allows for the recreation of mesh‐like mucus layer containing high level of mucus protective peptides and well‐developed epithelial junctional complexes. Furthermore, gastroprotection mechanisms against Helicobacter pylori (H. pylori) are successfully demonstrated in this system. Therefore, hsMPS represents a new in vitro tool for research where gastric mucosal defense mechanism is pivotal for developing therapeutic strategies

Exosome Precipitation by Ionic Strength Modulation: ExoPRISM

Extracellular vesicles (EVs) are emerging as crucial materials for precision theragnostic applications. However, current separation methods are time-consuming, costly, and not scalable and deliver limited yields or purity. Here, we present EV precipitation by ionic strength modulation (ExoPRISM), a simple, low-cost, user-friendly, and readily adaptable approach for separating EVs in high yields without compromising their biological functions. Adding an electrolyte solution to blood plasma in small increments generates the sequential precipitation of proteins and EVs, allowing for fractional separation of EVs using low-speed centrifugation. The coprecipitated electrolytes are easily washed away, and the entire EV separation and washing process takes less than an hour. This approach successfully separates EVs from a broad range of volumes and types of biological fluids, including culture medium, urine, plasma, and serum, showing promise as a robust tool for next-generation liquid biopsies and regenerative medicine. © 2023 The Authors. Published by American Chemical Society.11Nsciescopu