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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.
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Ciguatoxinas/aislamiento & purificación , Dinoflagelados/clasificación , Toxinas Marinas/aislamiento & purificación , Oxocinas/aislamiento & purificación , Animales , Australia , Línea Celular Tumoral , Cromatografía Liquida/métodos , Intoxicación por Ciguatera , Ciguatoxinas/toxicidad , Dinoflagelados/química , Humanos , Toxinas Marinas/toxicidad , Oxocinas/toxicidad , Espectrometría de Masas en Tándem , Clima TropicalRESUMEN
Corals are among the most active producers of dimethylsulfoniopropionate (DMSP), a key molecule in marine sulfur cycling, yet the specific physiological role of DMSP in corals remains elusive. Here, we examine the oxidative stress response of three coral species (Acropora millepora, Stylophora pistillata and Pocillopora damicornis) and explore the antioxidant role of DMSP and its breakdown products under short-term hyposalinity stress. Symbiont photosynthetic activity declined with hyposalinity exposure in all three reef-building corals. This corresponded with the upregulation of superoxide dismutase and glutathione in the animal host of all three species. For the symbiont component, there were differences in antioxidant regulation, demonstrating differential responses to oxidative stress between the Symbiodinium subclades. Of the three coral species investigated, only A. millepora provided any evidence of the role of DMSP in the oxidative stress response. Our study reveals variability in antioxidant regulation in corals and highlights the influence life-history traits, and the subcladal differences can have on coral physiology. Our data expand on the emerging understanding of the role of DMSP in coral stress regulation and emphasizes the importance of exploring both the host and symbiont responses for defining the threshold of the coral holobiont to hyposalinity stress.
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Antozoos/fisiología , Glutatión/metabolismo , Salinidad , Compuestos de Sulfonio/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Dinoflagelados/fisiología , Especificidad de la Especie , Estrés Fisiológico , SimbiosisRESUMEN
INTRODUCTION: The immunosuppressive tumor microenvironment is a major barrier for chemotherapy. Different chemosensitization approaches to reinstate immunological surveillance for cancers that are immune quiescent at the outset, have thus been devised. Cancer-specific ENOX2 expression is correlated with abnormal cell growth and has been proposed as a cellular target for anti-cancer activity. However, the potential effects of ENOX2 on the interaction between immune system and tumor cells remain elusive. OBJECTIVES: To understand the mechanisms by which tumor-intrinsic ENOX2-mediated alterations in anti-tumor activity of T-cells and response to chemotherapy. METHODS: In situ multiplexed immunohistochemistry with single cell and bulk RNA sequencing data from nasopharyngeal carcinoma (NPC) human tissues were used to define tumor phenotypes. Two NPC cell lines, with distinct ENOX2 expression, were used in a co-culture platform to study tumor-immune interactions between cancer cells/spheroids and T-cells. The effect of cisplatin treatment with ENOX2 inhibition by idronoxil (IDX) were tested in vitro and in vivo. Multi-parametric flow cytometry was used to characterize T-cell infiltrates in an NPC tumor humanized mouse model treated with combined treatment. RESULTS: NPC predominantly displayed an immune-excluded profile. This "cold-phenotype" was shown to exhibit higher ENOX2 expression and was associate with poorer progression-free survival (PFS). The therapeutic combination of IDX with cisplatin was effective in promoting CD8+ effector memory T cell (Tem) differentiation and mobilization. This Tem signature was highly cytotoxic, with Tem-mediated preferential lysis of higher ENOX2-expressing NPC cells. A combination-treated humanized mouse model showing dramatic shrinkage in tumors, were intra-tumoral Tem-enriched. CONCLUSION: Tumor-intrinsic ENOX2 expression is associated with tumor phenotype and PFS in NPC. Targeting ENOX2 with IDX and cisplatin impose qualitative control of T-cell response by preferentially increasing immune cells infiltration, Tem differentiation and tumor suppression. We suggest that ENOX2 inhibition may be a promising therapeutic strategy to enhance the effects of chemotherapy.
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Cisplatino , Neoplasias Nasofaríngeas , Humanos , Animales , Ratones , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Cisplatino/farmacología , Cisplatino/uso terapéutico , Células T de Memoria , Línea Celular Tumoral , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Microambiente TumoralRESUMEN
Microbes are sensitive indicators of estuarine processes because they respond rapidly to dynamic disturbance events. As most of the world's population lives in urban areas and climate change-related disturbance events are becoming more frequent, estuaries bounded by cities are experiencing increasing stressors, at the same time that their ecosystem services are required more than ever. Here, using a multidisciplinary approach, we determined the response of planktonic microbial assemblages in response to seasonality and a rainfall disturbance in an urban estuary bounded by Australia's largest city, Sydney. We used molecular barcoding (16S, 18S V4 rRNA) and microscopy-based identification to compare microbial assemblages at locations with differing characteristics and urbanisation histories. Across 142 samples, we identified 8,496 unique free-living bacterial zOTUs, 8,175 unique particle associated bacterial zOTUs, and 1,920 unique microbial eukaryotic zOTUs. Using microscopy, we identified only the top <10% abundant, larger eukaryotic taxa (>10 µm), however quantification was possible. The site with the greater history of anthropogenic impact showed a more even community of associated bacteria and eukaryotes, and a significant increase in dissolved inorganic nitrogen following rainfall, when compared to the more buffered site. This coincided with a reduced proportional abundance of Actinomarina and Synechococcus spp., a change in SAR 11 clades, and an increase in the eukaryotic microbial groups Dinophyceae, Mediophyceae and Bathyoccocaceae, including a temporary dominance of the harmful algal bloom dinoflagellate Prorocentrum cordatum (syn. P. minimum). Finally, a validated hydrodynamic model of the estuary supported these results, showing that the more highly urbanised and upstream location consistently experienced a higher magnitude of salinity reduction in response to rainfall events during the study period. The best abiotic variables to explain community dissimilarities between locations were TDP, PN, modelled temperature and salinity (r = 0.73) for the free living bacteria, TP for the associated bacteria (r = 0.43), and modelled temperature (r = 0.28) for the microbial eukaryotic communities. Overall, these results show that a minor disturbance such as a brief rainfall event can significantly shift the microbial assemblage of an anthropogenically impacted area within an urban estuary to a greater degree than a seasonal change, but may result in a lesser response to the same disturbance at a buffered, more oceanic influenced location. Fine scale research into the factors driving the response of microbial communities in urban estuaries to climate related disturbances will be necessary to understand and implement changes to maintain future estuarine ecosystem services.
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Diatomeas , Dinoflagelados , Ecosistema , Estuarios , Plancton , Océanos y Mares , Bacterias/genéticaRESUMEN
TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.
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COVID-19 , Interferón Tipo I , Animales , Ratones , Quinasa I-kappa B , Modelos Animales de Enfermedad , SARS-CoV-2 , InflamaciónRESUMEN
Testosterone is one of the androgenic steroid hormones, the consumption of which is considered doping in most sports. Here, we present powerful 3D sensing platforms using novel disc-ring microelectrode array devices and exploit them for the competitive immunosensing of testosterone. Each device contains a microelectrode array that consists of a large number of individual microdiscs and is used as the substrate for immunofunctionalization and assay performance. One micrometer above it, a second microelectrode array, this time consisting of microrings, is used as the working electrode for electrochemical monitoring. The physical separation of these two functions allows the incorporation of relatively thick biocomponent layers during immunofunctionalization of the microdiscs without negatively affecting electrochemical detection at the rings. Moreover, it permits electrochemical activation of the latter immediately before substrate addition and hence enables optimal electrode performance. The optimized assay showed a linear range between 0.01 and 10 ng/mL and a limit of detection of 12.5 pg/mL testosterone with detection times of 45 min.
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Doping en los Deportes/métodos , Técnicas Electroquímicas/métodos , Testosterona/análisis , Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoensayo/métodos , MicroelectrodosRESUMEN
Idronoxil has been the subject of more than 50 peer-reviewed publications over the last two decades. This isoflavone is an intriguing regulator of multiple signal transduction pathways, capable of causing a range of biological effects, including cell cycle arrest, apoptosis, an ability to stimulate the immune system, and inhibition of angiogenesis. These multifaceted actions suggest that idronoxil has the potential to synergize with, or complement, a wide range of cancer therapies. Whilst clinically tested in the past, idronoxil's journey was discontinued as a result of its low bioavailability in humans when administered either intravenously or orally, though strategies to overcome this issue are currently being explored. Here, we summarize the current literature regarding the key cellular targets of idronoxil and the mechanisms by which idronoxil exerts its anticancer effects, laying a new foundation toward giving this unique molecule a second chance of contributing to the future of cancer treatment.
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Antineoplásicos/uso terapéutico , Isoflavonas/uso terapéutico , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis , Ciclo Celular , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Experimentation at sea provides insight into which traits of ocean microbes are linked to performance in situ. Here we show distinct patterns in thermal tolerance of microbial phototrophs from adjacent water masses sampled in the south-west Pacific Ocean, determined using a fluorescent marker for reactive oxygen species (ROS). ROS content of pico-eukaryotes was assessed after 1, 5 and 25 h of incubation along a temperature gradient (15.6-32.1 °C). Pico-eukaryotes from the East Australian Current (EAC) had relatively constant ROS and showed greatest mortality after 25 h at 7 °C below ambient, whereas those from the Tasman Sea had elevated ROS in both warm and cool temperature extremes and greatest mortality at temperatures 6-10 °C above ambient, interpreted as the outcome of thermal stress. Tracking of water masses within an oceanographic circulation model showed populations had distinct thermal histories, with EAC pico-eukaryotes experiencing higher average temperatures for at least 1 week prior to sampling. While acclimatization and community assembly could both influence biological responses, this study clearly demonstrates that phenotypic divergence occurs along planktonic drift trajectories.
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Bacterias/aislamiento & purificación , Fotosíntesis , Agua de Mar/química , Agua de Mar/microbiología , Animales , Australia , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Calor , Océano Pacífico , Plancton , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , TemperaturaRESUMEN
This paper presents an immunosensing system to detect Escherichia coli and Salmonella based on electrochemical impedance spectroscopy at interdigitated electrode structures. Our results show the importance of good electrode design in the final detection limit. Four different structures have been fabricated and functionalized. Biotinylated polyclonal antibodies have been immobilized on neutravidin-coated chips, and BSA has been used to avoid nonspecific adsorption. The immunosensor may be said to be capacitive since it is that part of the impedance used to monitor the presence of bacteria in phosphate buffer solution samples. Detection limits around 10(4)-10(5) cells mL(-1) have been reached using chips featuring interdigitated structures of less than 10 microm wide and 1.5 mm long. In both cases, the detection limits of the corresponding ELISA assays, using the same antibodies, was 1 order of magnitude higher (10(5)-10(6) cells mL(-1)). The analysis time, including sensor preparation was less than 5 h.
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Técnicas Bacteriológicas/métodos , Técnicas Electroquímicas/métodos , Escherichia coli/aislamiento & purificación , Salmonella typhimurium/aislamiento & purificación , Especificidad de Anticuerpos , Técnicas Biosensibles , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/química , Microelectrodos , Salmonella typhimurium/química , Sensibilidad y EspecificidadRESUMEN
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.
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Técnicas Biosensibles/métodos , Escherichia coli K12/aislamiento & purificación , Oro , Técnicas para Inmunoenzimas/métodos , Proteínas/análisis , Adsorción , Anticuerpos Antibacterianos/inmunología , Escherichia coli K12/inmunologíaRESUMEN
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.
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Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/inmunología , Escherichia coli/inmunología , Escherichia coli/aislamiento & purificación , Anticuerpos/inmunología , Oro/químicaRESUMEN
Functionalisation of the sensing surface is a key factor in immunosensor fabrication as it allows target-selective capture and prevents nonspecific adsorption of undesired components. Gold immunofunctionalisation using self-assembled monolayers (SAM) has been widely exploited to this end for the detection of small targets. However, we recently demonstrated that this strategy fails when detecting whole bacteria cells (Baldrich et al., Anal Bioanal Chem 390:1557-1562, 2008). We now investigate different physisorption-based alternatives using E. coli as the target organism. Our results demonstrate that physisorption generates the appropriate substrate for the specific detection of bacteria on gold surfaces, providing detection limits down to 10(5) cells mL(-1) in an ELISA-type colorimetric assay. Additionally, surface coverage is highly reproducible when assayed by impedance spectroscopy and the inter- and intra-assay coefficients of variation are below 10-15% in all cases. These surfaces were stable, retained functionality and did not suffer from significant biomolecule desorption after 10 days storage in PBS at 37 degrees C, hence confirming physisorption as a cheap, simple and efficient strategy for the detection of bacteria.
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Bacterias/aislamiento & purificación , Adhesión Bacteriana , Técnicas Biosensibles/métodos , Oro/química , Inmunoensayo , Absorción , Técnicas Biosensibles/instrumentación , Ensayo de Inmunoadsorción Enzimática , Inmunoensayo/métodos , Reproducibilidad de los ResultadosRESUMEN
Two different bacteria gave different respiratory responses to the test analytes, tributyl tin (TBT) and cadmium as expressed by positive sigmoid responses by Halomonas sp. (slope, +1.71 [TBT]; +1.76 [Cd]) and negative sigmoid responses by Bacillus pumilis (slope, -1.06 [TBT]; -0.59 [Cd]). The EC50 values determined from Hill plots for the response of Halomonas sp. to the TBT and Cd were 1 and 8.5 mM, respectively, which were lower by a factor of 10 than the corresponding values for B. pumilis. With protoplasts of B. pumilis there was a major shift in the signal from sigmoid negative to positive with TBT (+1.35) but not Cd (-0.5), while the signals with the remaining protoplast-analyte combinations remained unchanged. For all four protoplast-analyte combinations the EC50 values were in the order of 10-100-fold lower than those for their whole cell counterparts. When other analytes were tested the protoplasts gave a similar response to tin as for TBT, but detected copper and 2,4-dichlorophenol with similar signal profiles to Cd and with lower sensitivity. The difference in signal and higher sensitivity of the two species protoplast system towards TBT/tin compared to the other analytes tested, suggests that it may feasible to develop this approach for the detection of tin residues.
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Bacillus , Técnicas Biosensibles , Halomonas , Protoplastos , Estaño/análisisRESUMEN
Ciguatera Fish Poisoning (CFP) is a tropical disease caused by the consumption of fish contaminated with ciguatoxins (CTXs). Currently, the only feasible prevention methods for CFP are to avoid the consumption of fish of certain species from some regions, avoid larger fish of certain species, or avoid all fish caught from specific regions. Here, we quantified levels of P-CTX-1B in Spanish Mackerel (Scomberomorus commerson), which is the main fish species that causes CFP in New South Wales and Queensland, Australia, using LC-MS detection against a toxin standard. We found detectable P-CTX-1B in both flesh and liver tissues in fish from New South Wales (n = 71, 1.4% prevalence rate, with a confidence interval of 1%-4%, and 7% prevalence, 1%-12%, in flesh and liver, respectively). In the small sample of fish from Queensland, there was a 46% prevalence (19-73%, n = 13). Toxin levels found were 0.13 µg kg-1 to <0.1 µg kg-1 in flesh, and 1.39 µg kg-1 to <0.4 µg kg-1 in liver, indicating that liver tissue had a significantly higher concentration (â¼5 fold) of P-CTX-1B. No apparent relationship was observed between the length or weight of S. commerson and the detection of P-CTX-1B in this study. Footnote.
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Bacteria from the genus Vibrio 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 Vibrio genus. The assay was optimized on 12 diverse Vibrio strains, including species that have implications for aquaculture industries, reaching detection limits between 7×10(3) to 3×10(4) 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.
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Acuicultura/métodos , Ambiente , Vibrio/aislamiento & purificación , Microbiología del Agua , Animales , HumanosRESUMEN
We report a novel electrochemical method for the rapid detection of the parasitic protozoan, Cryptosporidium parvum. An antibody-based capture format was transferred onto screen-printed electrodes and the presence of horseradish peroxidase-labelled antibodies binding to the oocysts was potentiometrically detected. This method allowed the detection of 5 × 10(2)Cryptosporidium oocysts per mL in 60 min.
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Cryptosporidium parvum/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Oocistos , Potenciometría/métodos , Anticuerpos/química , Cryptosporidium parvum/genética , ElectrodosRESUMEN
This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and amperometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H(2)O(2)) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is amperometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 10(2)-10(8) cell ml(-1), with a limit of detection of 55 cells ml(-1) in PBS, without pre-enrichment steps. Furthermore, 100 cells ml(-1) could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.
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Bacterias/aislamiento & purificación , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Técnicas Analíticas Microfluídicas , Electrodos , Escherichia coli/aislamiento & purificación , MagnetismoRESUMEN
Two simple methodologies are compared for the detection of faecal contamination in water using amperometry at gold interdigitated microelectrodes. They rely on the detection of ß-galactosidase (ß-gal) by redox cycling amperometry of the p-aminophenol (PAP) produced by the enzyme from the 4-aminophenyl ß-d-galactopyranoside (PAPG) substrate. The use of phages as specific agents for the release of the bacteria-enclosed enzyme allowed the detection of 6×10(5) CFU mL(-1)Escherichia coli in 2 h without any pre-enrichment or preconcentration steps. Better limits of detection were achieved for the second strategy in the absence of phages. In this case, bacteria were enriched in the presence of both ß-d-1-thiogalactopyranoside (IPTG) and substrate but in the absence of phages. Under such experimental conditions, 5×10(4) CFU mL(-1) E. coli could be detected after 2 h of incubation, while 7 h of incubation were enough to detect down to 10 CFU mL(-1) in river water samples. This represents a straightforward one-step method for the detection of faecal contamination that can be conducted in a single working day with minimal sample manipulation by the user.
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Técnicas Biosensibles/métodos , Enterobacteriaceae/aislamiento & purificación , Ríos/química , Microbiología del Agua , Abastecimiento de Agua/análisis , beta-Galactosidasa/análisis , Aminofenoles/análisis , Aminofenoles/química , Electroquímica , Enterobacteriaceae/enzimología , Glucósidos/metabolismo , Límite de Detección , Microelectrodos , Oxidación-Reducción , Factores de Tiempo , Abastecimiento de Agua/normas , beta-Galactosidasa/metabolismoRESUMEN
Here a novel electrochemical method for the rapid detection of anti-HIV antibodies in serum is presented. The novelty lies in the combination of allosteric enzymes and coulometry to yield a fast, simple and reliable HIV diagnostic method. We have used a previously developed beta-galactosidase enzyme that is efficiently activated by anti-HIV antibodies directed against a major B-cell epitope of the gp41 glycoprotein. When these antibodies bind the enzyme, the 3D conformation changes positively affecting the performance of the active site and, consequently, the enzyme activity is stimulated. Using 4-aminophenyl beta-D-galactopyranoside (PAPG) as substrate yields p-aminophenol (PAP), which is reversibly oxidised at a very mild potential, ca. 0.37 V vs. Ag/AgCl over a range of electrode materials within the working pH range of beta-galactosidase. In the present case, photolithographically produced microelectrode arrays resulted in a detection limit of 4 microM for 4-aminophenol (PAP). The presence of anti-HIV antibodies results in enzyme activity increases above 50% which, combined with the sensitivity and response time afforded by the microelectrode arrays, allowed for the diagnosis of HIV in sera samples within an hour.