Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

International audienceToday, rapid detection and identification of bacteria in microbiological diagnosis is a major issue. Reference methods usually rely on growth of microorganisms, with the drawback of lengthy time-to-result. The method provides global information on a clonal population that is known to be inhomogeneous relative to metabolic states and activities. Therefore, there may be a significant advantage of methods that allow characterisation of individual bacteria from a large population, both for test time reduction and the clinical value of the characterisation. We report here a method for rapid detection and real-time monitoring of the metabolic activities of single bacteria. Water-in-oil emulsions were used to encapsulate single Escherichia coli cells into picolitre (pL)-sized microreactor droplets. The glucuronidase activity in each droplet was monitored using the fluorogenic reporter molecule MUG (4-methylumbelliferyl- - d-glucuronide) coupled to time-lapse fluorescence imaging of the droplets. Such bacterial confinement provides several major advantages. (1) Enzymatic activities of a large number of single bacterium-containing droplet could be monitored simultaneously, allowing the full characterisation of metabolic heterogeneity in a clonal population. We monitored glucuronidase enzymatic activity and growth over ∼200 single bacteria over a 24-h period. (2) Micro-confinement of cells in small volumes allows rapid accumulation of the fluorescent metabolite, hence decreasing the detection time. Independent of the initial concentration of bacteria in the sample, detection of the presence of bacteria could be achieved in less than 2 h. (3) Considering the random distribution of bacteria in droplets, this method allowed rapid and reliable enumeration of bacteria in the initial sample. Overall, the results of this study showed that confinement of bacterial cells increased the effective concentration of fluorescent metabolites leading to rapid (2 h) detection of the fluorescent metabolites, thus significantly reducing time to numeration

Antimicrobial peptide arrays for wide spectrum sensing of pathogenic bacteria

International audience10 Fast detection of bacteria in samples presumed to be un-contaminated, such as blood, is of great 11 importance. Indeed, rapid diagnosis allows the setup of appropriate antibiotic treatment. Besides clinical 12 issues, there are many other domains, such as food processing or drug manufacturing, where the strict 13 absence of any bacteria has to be assessed. Because the bacterial load found in most contaminated 14 samples is often below the limit of detection for currently validated assays, a preliminary enrichment step 15 is required to allow bacterial multiplication before proceeding to the analysis step, whatever it might be-16 cultural, immunological or molecular methods. In this study, we describe the use of a biosensor for single-17 step bacteria detection. The whole analysis is performed in less than 20 hours, during the growth phase of 18 the microorganisms , using an array of antimicrobial peptides (AMPs) coupled with a surface plasmon 19 resonance imager (SPRI). A wide range of bacterial strains are assayed, showing differentiated affinity 20 patterns with the immobilized peptides, which are confirmed by multivariate analysis. This work 21 establishes the evidence that antimicrobial peptides, mostly used so far in the antibiotic drug industry, are 22 suited for the wide-spectrum detection of unknown bacteria in samples, even at very low initial loads. 23 Moreover, the small set of AMPs that were assayed provided a specific affinity profile for each pathogen, 24 as confirmed by multivariate analyses. Furthermore, this work opens up the possibility of applying this 25 method in more complex and relevant samples such as foodstuff, urine or blood. 26 27 2

SPR imaging of peptide micro-arrays for wide spectrum sensing of pathogenic bacteria

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Biomimetic optoelectronic nose for analysis of volatile organic compounds

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Recent Advances on Peptide-Based Biosensors and Electronic Noses for Foodborne Pathogen Detection

International audienceFoodborne pathogens present a serious issue around the world due to the remarkably high number of illnesses they cause every year. In an effort to narrow the gap between monitoring needs and currently implemented classical detection methodologies, the last decades have seen an increased development of highly accurate and reliable biosensors. Peptides as recognition biomolecules have been explored to develop biosensors that combine simple sample preparation and enhanced detection of bacterial pathogens in food. This review first focuses on the selection strategies for the design and screening of sensitive peptide bioreceptors, such as the isolation of natural antimicrobial peptides (AMPs) from living organisms, the screening of peptides by phage display and the use of in silico tools. Subsequently, an overview on the state-of-the-art techniques in the development of peptide-based biosensors for foodborne pathogen detection based on various transduction systems was given. Additionally, limitations in classical detection strategies have led to the development of innovative approaches for food monitoring, such as electronic noses, as promising alternatives. The use of peptide receptors in electronic noses is a growing field and the recent advances of such systems for foodborne pathogen detection are presented. All these biosensors and electronic noses are promising alternatives for the pathogen detection with high sensitivity, low cost and rapid response, and some of them are potential portable devices for on-site analyses

An Overview of Artificial Olfaction Systems with a Focus on Surface Plasmon Resonance for the Analysis of Volatile Organic Compounds

International audienceThe last three decades have witnessed an increasing demand for novel analytical tools for the analysis of gases including odorants and volatile organic compounds (VOCs) in various domains. Traditional techniques such as gas chromatography coupled with mass spectrometry, although very efficient, present several drawbacks. Such a context has incited the research and industrial communities to work on the development of alternative technologies such as artificial olfaction systems, including gas sensors, olfactory biosensors and electronic noses (eNs). A wide variety of these systems have been designed using chemiresistive, electrochemical, acoustic or optical transducers. Among optical transduction systems, surface plasmon resonance (SPR) has been extensively studied thanks to its attractive features (high sensitivity, label free, real-time measurements). In this paper, we present an overview of the advances in the development of artificial olfaction systems with a focus on their development based on propagating SPR with different coupling configurations, including prism coupler, wave guide, and grating

Surface plasmon resonance imaging based on peptide and HpDNA for sensing volatile organic compounds

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Screening of specific aptamers for Bacillus cereus by Surface Plasmon Resonance imaging (SPRi)

International audienceBacillus cereus is a food poisoning bacterium present in dairy products. To detect it, antibody based biosensors are efficient but available antibodies are expensive and may cross-react with other Bacillus species. Aptamers are a good alternative to these issues. Herein, seven aptamer sequences derived from the literature (1,2) are compared and their specificity to B. cereus is assessed versus Escherichia coli and Bacillus subtilis by Surface Plasmon Resonance imaging (SPRi)

Screening of specific aptamers for Bacillus cereus by Surface Plasmon Resonance imaging (SPRi)

International audienceBacillus cereus is a food poisoning bacterium present in dairy products. To detect it, antibody based biosensors are efficient but available antibodies are expensive and may cross-react with other Bacillus species. Aptamers are a good alternative to these issues. Herein, seven aptamer sequences derived from the literature (1,2) are compared and their specificity to B. cereus is assessed versus Escherichia coli and Bacillus subtilis by Surface Plasmon Resonance imaging (SPRi)