Biosensor para la detección de anticuerpos anti-VIH

Biosensor para la detección de anticuerpos anti-VIH. En la presente invención se provee un biosensor capaz de detectar anticuerpos anti-VIH en una muestra biológica, basado en la utilización combinada de una enzima alostérica, modificada genéticamente, y una matriz con redes de microelectrodos.Consejo Superior de Investigaciones Científicas (España), Universidad Autónoma de BarcelonaA1 Solicitud de patente con informe sobre el estado de la técnic

Colorimetric Detection of Caspase 3 Activity and Reactive Oxygen Derivatives: Potential Early Indicators of Thermal Stress in Corals

There is an urgent need to develop and implement rapid assessments of coral health to allow effective adaptive management in response to coastal development and global change. There is now increasing evidence that activation of caspase-dependent apoptosis plays a key role during coral bleaching and subsequent mortality. In this study, a "clinical" approach was used to assess coral health by measuring the activity of caspase 3 using a commercial kit. This method was first applied while inducing thermal bleaching in two coral species, Acropora millepora and Pocillopora damicornis. The latter species was then chosen to undergo further studies combining the detection of oxidative stress-related compounds (catalase activity and glutathione concentrations) as well as caspase activity during both stress and recovery phases. Zooxanthellae photosystem II (PSII) efficiency and cell density were measured in parallel to assess symbiont health. Our results demonstrate that the increased caspase 3 activity in the coral host could be detected before observing any significant decrease in the photochemical efficiency of PSII in the algal symbionts and/or their expulsion from the host. This study highlights the potential of host caspase 3 and reactive oxygen species scavenging activities as early indicators of stress in individual coral colonies

Self-assembled monolayers as a base for immunofunctionalisation: unequal performance for protein and bacteria detection

6 pages, 3 figures.-- PMID: 18256810 [PubMed].-- Printed version published Mar 2008.Biosensor development strongly depends on the optimisation of surface functionalisation strategies. When gold surfaces are considered, immunofunctionalisation by modification of self-assembled monolayers (SAMs) is one of the preferred approaches. In this respect, SAM-based antibody (Ab) incorporation has shown better performance than Ab physisorption for the detection of proteins and small targets. Reports on bacteria detection are less frequent. In this work, we assess the performance of various SAM-based gold immunofunctionalisation strategies, currently applied to protein detection, in the field of bacteria determination. We present the results for Ab chemical conjugation on mercaptopropanoic acid and mercaptoundecanoic acid SAMs, as well as on a dextranized cysteamine SAM. All the modified surfaces studied were shown to be appropriate for the direct detection of an enzyme-labelled protein, but none succeeded in detecting a bacterial target in a sandwich assay format. Conversely, gold functionalised by Ab physisorption allowed E. coli detection when a sandwich enzyme-linked assay was carried out. The implications of bacteria size and wall complexity are discussed. These results indicate that immunofunctionalisation strategies appropriate for protein detection are not necessarily transferable to work with more complex targets such as bacteria. In this respect, Ab physisorption appears to be a suitable alternative to SAM-based gold functionalisation for bacteria detection.This work has been funded in the context of the project MICROBACTOMETER (TEC2006-13109-C03-02/MIC) from the Spanish Ministry of Education. Eva Baldrich and F. Javier del Campo are respectively supported by an I3P fellowship from the Consejo Superior de Investigaciones Científicas (CSIC, Spain) and a Ramón y Cajal fellowship from the Spanish Ministry of Education.Peer reviewe

Gold immuno-functionalisation via self-assembled monolayers: Study of critical parameters and comparative performance for protein and bacteria detection

10 pages, 6 figures, 1 table.-- PMID: 18534611 [PubMed].-- Printed version published Jul 31, 2008.Surface functionalisation is of extreme importance in assay and biosensor development because it ensures the selective capture and detection of the targets of interest. In the present report, we compare the performance of several gold functionalisation strategies/chemistries, based on SAM self-assembly and Ab conjugation, for protein and bacteria detection.The first part of the work summarises the optimisation of the various protocols considered. Their efficiency was initially evaluated in terms of reduction of biomolecule non-specific adsorption and specific detection competence impairment, using as a model-target an enzyme-labelled protein. With this purpose, the effect of several parameters, such as thiomolecule length and concentration, self-assembly time and temperature, polymer incorporation, or Ab conjugation strategy was determined. The three best performing strategies consisted of antibody (Ab) conjugation to self-assembled monolayers (SAM) containing mercaptoundecanoic acid alone, or conjugated to either long-chain hydrophilic diamines or CM-dextran. In the three cases, results demonstrated that Abs had been successfully incorporated and remained functional for protein detection. Nevertheless, as showed in the second part of the work, we demonstrate for the first time that these chemistries can be inadequate for bacteria detection. The possible reasons and implications will be discussed. Ab physisorption is proposed as a cost-effective gold immuno-functionalisation strategy alternative to SAM-based Ab incorporation for bacteria detection.This work has been funded in the context of the project MICROBACTOMETER (TEC2006-13109-C03-02/MIC) from the Spanish Ministry of Education. Eva Baldrich is supported by an I3P fellowship from the Consejo Superior de Investigaciones Científicas (CSIC, Spain). Olivier Laczka and F. Javier del Campo are respectively supported by FP1 and a Ramón y Cajal fellowship from the Spanish Ministry of Education.Peer reviewe

Gold immuno-functionalisation via self-assembled monolayers: Study of critical parameters and comparative performance for protein and bacteria detection

10 pages, 6 figures, 1 table.-- PMID: 18534611 [PubMed].-- Printed version published Jul 31, 2008.Surface functionalisation is of extreme importance in assay and biosensor development because it ensures the selective capture and detection of the targets of interest. In the present report, we compare the performance of several gold functionalisation strategies/chemistries, based on SAM self-assembly and Ab conjugation, for protein and bacteria detection.The first part of the work summarises the optimisation of the various protocols considered. Their efficiency was initially evaluated in terms of reduction of biomolecule non-specific adsorption and specific detection competence impairment, using as a model-target an enzyme-labelled protein. With this purpose, the effect of several parameters, such as thiomolecule length and concentration, self-assembly time and temperature, polymer incorporation, or Ab conjugation strategy was determined. The three best performing strategies consisted of antibody (Ab) conjugation to self-assembled monolayers (SAM) containing mercaptoundecanoic acid alone, or conjugated to either long-chain hydrophilic diamines or CM-dextran. In the three cases, results demonstrated that Abs had been successfully incorporated and remained functional for protein detection. Nevertheless, as showed in the second part of the work, we demonstrate for the first time that these chemistries can be inadequate for bacteria detection. The possible reasons and implications will be discussed. Ab physisorption is proposed as a cost-effective gold immuno-functionalisation strategy alternative to SAM-based Ab incorporation for bacteria detection.This work has been funded in the context of the project MICROBACTOMETER (TEC2006-13109-C03-02/MIC) from the Spanish Ministry of Education. Eva Baldrich is supported by an I3P fellowship from the Consejo Superior de Investigaciones Científicas (CSIC, Spain). Olivier Laczka and F. Javier del Campo are respectively supported by FP1 and a Ramón y Cajal fellowship from the Spanish Ministry of Education.Peer reviewe

Biosensor para la detección de anticuerpos anti-VIH

[EN] In the present invention, provision is made for a biosensor capable of detecting anti-HIV antibodies in a biological sample, based on the combined use of a genetically modified allosteric enzyme and a matrix with microelectrode networks. One of the advantages of this biosensor is the high sensitivity thereof, the simplicity thereof in terms of detection and the portability thereof.[ES] En Ia presente invención se provee un biosensor capaz de detectar anticuerpos anti-VIH en una muestra biológica, basado en la utilización combinada de una enzima alostérica, modificada genéticamente, y una matriz con redes de microelectrodos. Una de las ventajas de este biosensor es su alta sensibilidad, su sencillez en la detección y su portabilidad.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Autónoma de Barcelona (UAB)A1 Solicitud de patentes con informe sobre el estado de la técnic

Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins

Surface Immuno-Functionalisation for the Capture and Detection of <i>Vibrio</i> Species in the Marine Environment: A New Management Tool for Industrial Facilities

<div><p>Bacteria from the genus <i>Vibrio</i> are a common and environmentally important group of bacteria within coastal environments and include species pathogenic to aquaculture organisms. Their distribution and abundance are linked to specific environmental parameters, including temperature, salinity and nutrient enrichment. Accurate and efficient detection of Vibrios in environmental samples provides a potential important indicator of overall ecosystem health while also allowing rapid management responses for species pathogenic to humans or species implicated in disease of economically important aquacultured fish and invertebrates. In this study, we developed a surface immuno-functionalisation protocol, based on an avidin-biotin type covalent binding strategy, allowing specific sandwich-type detection of bacteria from the <i>Vibrio</i> genus. The assay was optimized on 12 diverse <i>Vibrio</i> strains, including species that have implications for aquaculture industries, reaching detection limits between 7×10³ to 3×10⁴ cells mL⁻¹. Current techniques for the detection of total Vibrios rely on laborious or inefficient analyses resulting in delayed management decisions. This work represents a novel approach for a rapid, accurate, sensitive and robust tool for quantifying Vibrios directly in industrial systems and in the environment, thereby facilitating rapid management responses.</p></div

Affinity of the different <i>Vibrio</i> strains and commercial positive control to the functionalised surface using optimised conditions.

<p>Absorbance signals obtained after a 30 min cell capture step on the functionalised surface and a 30 min detection step using a 1/1000 dilution of horseradish peroxidase anti-<i>Vibrio</i> antibody (HRP-α<i>Vib</i> Pab).</p