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In the context of bacteriophage (phage) therapy, there is an urgent need for a method permitting multiplexed, parallel phage susceptibility testing (PST) prior to the formulation of personalized phage cocktails for administration to patients suffering from antimicrobial-resistant bacterial infections. Methods based on surface plasmon resonance imaging (SPRi) and phase imaging were demonstrated as candidates for very rapid (<2 h) PST in the broth phase. Biosensing layers composed of arrays of phages 44AHJD, P68, and gh-1 were covalently immobilized on the surface of an SPRi prism and exposed to liquid culture of either Pseudomonas putida or methicillin-resistant Staphylococcus aureus (i.e., either the phages' host or non-host bacteria). Monitoring of reflectivity reveals susceptibility of the challenge bacteria to the immobilized phage strains. Investigation of phase imaging of lytic replication of gh-1 demonstrates PST at the single-cell scale, without requiring phage immobilization. SPRi sensorgrams show that on-target regions increase in reflectivity more slowly, stabilizing later and to a lower level compared to off-target regions. Phage susceptibility can be revealed in as little as 30 min in both the SPRi and phase imaging methods. [ABSTRACT FROM AUTHOR]

Loop-mediated isothermal amplification (LAMP) is an exponential amplification method of DNA strands that is more and more used for its high performances. Thanks to its high sensitivity and selectivity, LAMP found numerous applications from the detection of pathogens or viruses through their genome amplification to its incorporation as an amplification strategy in protein or miRNA biomarker quantification. The LAMP method is composed of two stages: the first one consists in the transformation of the DNA strands into dumbbell structures formed of two stems and loops thanks to four primers; then, in the second stage, only two primers are required to amplify the dumbbells exponentially in numerous hairpins of increasing lengths. In this paper, we propose a theoretical framework to analyze the kinetics of the second stage of LAMP, the isothermal dumbbell exponential amplification (IDEA) as function of the physico-chemical parameters of the amplification reaction. Dedicated experiments validate the models. We believe these results may help the optimization of LAMP performances by reducing the number of experiments necessary to find the best parameters. [ABSTRACT FROM AUTHOR]

The development of sensitive methods for in situ detection of biomarkers is a real challenge to bring medical diagnosis a step forward. The proof-of-concept of a remote multiplexed biomolecular interaction detection through a plasmonic optical fiber bundle is demonstrated here. The strategy relies on a fiber optic biosensor designed from a 300 µm diameter bundle composed of 6000 individual optical fibers. When appropriately etched and metallized, each optical fiber exhibits specific plasmonic properties. The surface plasmon resonance phenomenon occurring at the surface of each fiber enables to measure biomolecular interactions, through the changes of the retro-reflected light intensity due to light/plasmon coupling variations. The functionalization of the microstructured bundle by multiple protein probes was performed using new polymeric 3D-printed microcantilevers. Such soft cantilevers allow for immobilizing the probes in micro spots, without damaging the optical microstructures nor the gold layer. We show here the potential of this device to perform the multiplexed detection of two different antibodies with limits of detection down to a few tenths of nanomoles per liter. This tool, adapted for multiparametric, real-time, and label free monitoring is minimally invasive and could then provide a useful platform for in vivo targeted molecular analysis. [ABSTRACT FROM AUTHOR]

The identification and characterization, at the cellular level, of cytokine productions present a high interest for both fundamental research and clinical studies. However, the majority of techniques currently available (ELISA, ELISpot, flow cytometry, etc.) have several shortcomings including, notably, the assessment of several cytokines in relation to individual secreting cells and the monitoring of living cell responses for a long incubation time. In the present work, we describe a system composed of a microfluidic platform coupled with an antibody microarray chip for continuous SPR imaging and immunofluorescence analysis of cytokines (IL-2 and IFN-γ) secreted by T-Lymphocytes, specifically, and stably captured on the biochip under flow upon continued long-term on-chip culture (more than 24 h). [ABSTRACT FROM AUTHOR]

The detection of small molecules by biosensors remains a challenge for diagnostics in many areas like pharmacology, environment or homeland security. The main difficulty comes from both the low molecular weight and low concentrations of most targets, which generally requires an indirect detection with an amplification or a sandwich procedure. In this study, we combine both strategies as the amplification of Surface Plasmon Resonance imaging (SPRi) signal is obtained by the use of gold nanoparticles and the sequence engineering of split-aptamers, short oligonucleotides strands with strong affinity towards small targets, allows for a sandwich structure. Combining those two strategies, we obtained state-of-the-art results in the limit of detection (LOD = 50 nM) with the model target adenosine. Furthermore, the SPRi detection led on aptamer microarrays paves the way for potential multi-target detections thanks to the multi-probe imaging approach. [ABSTRACT FROM AUTHOR]

Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak. [ABSTRACT FROM AUTHOR]

Bacterial resistance is becoming a global issue due to its rapid growth. Potential new drugs as antimicrobial peptides (AMPs) are considered for several decades as promising candidates to circumvent this threat. Nonetheless, AMPs have also been used more recently in other settings such as molecular probes grafted on biosensors able to detect whole bacteria. Rapid, reliable and cost-efficient diagnostic tools for bacterial infection could prevent the spread of the pathogen from the earliest stages. Biosensors based on AMPs would enable easy monitoring of potentially infected samples, thanks to their powerful versatility and integrability in pre-existent settings. AMPs, which show a broad spectrum of interactions with bacterial membranes, can be tailored in order to design ubiquitous biosensors easily adaptable to clinical settings. This review aims to focus on the state of the art of AMPs used as the recognition elements of whole bacteria in label-free biosensors with a particular focus on the characteristics obtained in terms of threshold, volume of sample analysable and medium, in order to assess their workability in real-world applications. [ABSTRACT FROM AUTHOR]

This review summarizes recent advances in micro- and nanopore technologies with a focus on the functionalization of pores using a promising method named contactless electro-functionalization (CLEF). CLEF enables the localized grafting of electroactive entities onto the inner wall of a micro- or nano-sized pore in a solid-state silicon/silicon oxide membrane. A voltage or electrical current applied across the pore induces the surface functionalization by electroactive entities exclusively on the inside pore wall, which is a significant improvement over existing methods. CLEF's mechanism is based on the polarization of a sandwich-like silicon/silicon oxide membrane, creating electronic pathways between the core silicon and the electrolyte. Correlation between numerical simulations and experiments have validated this hypothesis. CLEF-induced micro- and nanopores functionalized with antibodies or oligonucleotides were successfully used for the detection and identification of cells and are promising sensitive biosensors. This technology could soon be successfully applied to planar configurations of pores, such as restrictions in microfluidic channels. [ABSTRACT FROM AUTHOR]