RESUMEN
Improving the sensitivity of plasmonic optical fiber sensors constitutes a major challenge as it could significantly enhance their sensing capabilities for the label-free detection of biomolecular interactions or chemical compounds. While many efforts focus on developing more sensitive structures, we present here how the sensitivity of a sensor can be significantly enhanced by improving the light analysis. Contrary to the common approach where the global intensity of the light coming from the core is averaged, our approach is based on the full analysis of the retro-reflected intensity distribution that evolves with the refractive index of the medium being analyzed. Thanks to this original and simple approach, the refractive index sensitivity of a plasmonic optical fiber sensor used in reflection mode was enhanced by a factor of 25 compared to the standard method. The reported approach opens exciting perspectives for improving the remote detection as well as for developing new sensing strategies.
RESUMEN
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.
Asunto(s)
Técnicas Biosensibles/métodos , Fibras Ópticas , Resonancia por Plasmón de Superficie/métodos , Animales , Anticuerpos/análisis , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Oro/química , Límite de Detección , Ratas , Resonancia por Plasmón de Superficie/instrumentación , Propiedades de SuperficieRESUMEN
Remote detection by surface plasmon resonance (SPR) is demonstrated through microstructured optical arrays of conical nanotips or micropillars. Both geometries were fabricated by controlled wet chemical etching of bundles comprising several thousands of individual optical fibers. Their surface was coated by a thin gold layer in order to confer SPR properties. The sensitivity and resolution of both shapes were evaluated as a function of global optical index changes in remote detection mode performed by imaging through the etched optical fiber bundle itself. With optimized geometry of micropillar arrays, resolution was increased up to 10-4 refractive index units. The gold-coated micropillar arrays were functionalized with DNA and were able to monitor remotely the kinetics of DNA hybridization with complementary strands. We demonstrate for the first time highly parallel remote SPR detection of DNA via microstructured optical arrays. The obtained SPR sensitivity combined with the remote intrinsic properties of the optical fiber bundles should find promising applications in biosensing, remote SPR imaging, a lab-on-fiber platform dedicated to biomolecular analysis, and in vivo endoscopic diagnosis. Graphical abstract We present a single fabrication step to structure simultaneously all the individual cores of an optical fiber bundle composed of thousands of fibers. The resulting sensor is optimized for reflection mode (compatible with in vivo applications) and is used to perform for the first time highly parallel remote SPR detection of DNA via several thousands of individual optical fiber SPR sensors paving the way for multiplexed biological detection.
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ADN/análisis , Hibridación de Ácido Nucleico , Fibras Ópticas , Resonancia por Plasmón de Superficie/instrumentación , Diseño de Equipo , Tecnología de Fibra Óptica/instrumentación , Oro/química , Ácidos Nucleicos Inmovilizados/química , RefractometríaRESUMEN
BACKGROUND: Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NF-κB dependent signals which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, we created and screened a focused RNA interference library on Drosophila NF-κB-like pathways Toll and Imd in cultured S2 cells, and further analysed the function of selected genes in vivo. RESULTS: We report here that USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NF-κB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. We further show that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments. CONCLUSION: Our work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular , Drosophila/inmunología , Drosophila/microbiología , Bacterias Gramnegativas , Bacterias Grampositivas , Transducción de Señal , Receptores Toll-Like/metabolismo , UbiquitinaciónRESUMEN
This review takes stock of the various optical fiber-based biosensors that could be used for in vivo applications. We discuss the characteristics that biosensors must have to be suitable for such applications and the corresponding transduction modes. In particular, we focus on optical fiber biosensors based on fluorescence, evanescent wave, plasmonics, interferometry, and Raman phenomenon. The operational principles, implemented solutions, and performances are described and debated. The different sensing configurations, such as the side- and tip-based fiber biosensors, are illustrated, and their adaptation for in vivo measurements is discussed. The required implementation of multiplexed biosensing on optical fibers is shown. In particular, the use of multi-fiber assemblies, one of the most optimal configurations for multiplexed detection, is discussed. Different possibilities for multiple localized functionalizations on optical fibers are presented. A final section is devoted to the practical in vivo use of fiber-based biosensors, covering regulatory, sterilization, and packaging aspects. Finally, the trends and required improvements in this promising and emerging field are analyzed and discussed.
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Técnicas Biosensibles , Fibras Ópticas , InterferometríaRESUMEN
In this work, we introduce a polymer version of a previously developed silicon MEMS drop deposition tool for surface functionalization that consists of a microcantilever integrating an open fluidic channel and a reservoir. The device is fabricated by laser stereolithography, which offers the advantages of low-cost and fast prototyping. Additionally, thanks to the ability to process multiple materials, a magnetic base is incorporated into the cantilever for convenient handling and attachment to the holder of a robotized stage used for spotting. Droplets with diameters ranging from â¼50 µm to â¼300 µm are printed upon direct contact of the cantilever tip with the surface to pattern. Liquid loading is achieved by fully immersing the cantilever into a reservoir drop, where a single load results in the deposition of more than 200 droplets. The influences of the size and shape of the cantilever tip and the reservoir on the printing outcome are studied. As a proof-of-concept of the biofunctionalization capability of this 3D printed droplet dispenser, microarrays of oligonucleotides and antibodies displaying high specificity and no cross-contamination are fabricated, and droplets are deposited at the tip of an optical fiber bundle.
RESUMEN
This work proposes a miniaturized system able to perform multiple cell capture followed by cell-type selective release from a biochip surface. Unlabelled lymphocytes were first specifically captured onto a DNA array by antibody-DNA conjugates. The immobilized cells were subsequently released under spatiotemporal control within local heating generated by intense Surface Plasmon Resonance (SPR) produced by laser illumination.
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Anticuerpos/química , Linfocitos B/química , ADN/química , Resonancia por Plasmón de Superficie , Linfocitos T/citología , Análisis de Matrices Tisulares , Animales , Linfocitos B/metabolismo , Células Inmovilizadas/citología , Células Inmovilizadas/metabolismo , Ratones , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/métodos , Linfocitos T/metabolismo , Análisis de Matrices Tisulares/instrumentación , Análisis de Matrices Tisulares/métodosRESUMEN
Ubiquitin proteases remove ubiquitin monomers or polymers to modify the stability or activity of proteins and thereby serve as key regulators of signal transduction. Here, we describe the function of the Drosophila ubiquitin-specific protease 36 (dUSP36) in negative regulation of the immune deficiency (IMD) pathway controlled by the IMD protein. Overexpression of catalytically active dUSP36 ubiquitin protease suppresses fly immunity against Gram-negative pathogens. Conversely, silencing dUsp36 provokes IMD-dependent constitutive activation of IMD-downstream Jun kinase and NF-kappaB signaling pathways but not of the Toll pathway. This deregulation is lost in axenic flies, indicating that dUSP36 prevents constitutive immune signal activation by commensal bacteria. dUSP36 interacts with IMD and prevents K63-polyubiquitinated IMD accumulation while promoting IMD degradation in vivo. Blocking the proteasome in dUsp36-expressing S2 cells increases K48-polyubiquitinated IMD and prevents its degradation. Our findings identify dUSP36 as a repressor whose IMD deubiquitination activity prevents nonspecific activation of innate immune signaling.
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Proteínas de Drosophila/fisiología , Drosophila/inmunología , Endopeptidasas/fisiología , Regulación de la Expresión Génica , Transducción de Señal , Animales , Dosificación de Gen , Silenciador del Gen , Vida Libre de Gérmenes/inmunología , Bacterias Gramnegativas/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , FN-kappa B/biosíntesis , Mapeo de Interacción de ProteínasRESUMEN
Nonaspanins are characterised by a large N-terminal extracellular domain and nine putative transmembrane domains. This evolutionarily conserved family comprises three members in Dictyostelium discoideum (Phg1A, Phg1B and Phg1C) and Drosophila melanogaster, and four in mammals (TM9SF1-TM9SF4), the function of which is essentially unknown. Genetic studies in Dictyostelium demonstrated that Phg1A is required for cell adhesion and phagocytosis. We created Phg1A/TM9SF4-null mutant flies and showed that they were sensitive to pathogenic Gram-negative, but not Gram-positive, bacteria. This increased sensitivity was not due to impaired Toll or Imd signalling, but rather to a defective cellular immune response. TM9SF4-null larval macrophages phagocytosed Gram-negative E. coli inefficiently, although Gram-positive S. aureus were phagocytosed normally. Mutant larvae also had a decreased wasp egg encapsulation rate, a process requiring haemocyte-dependent adhesion to parasitoids. Defective cellular immunity was coupled to morphological and adhesion defects in mutant larval haemocytes, which had an abnormal actin cytoskeleton. TM9SF4, and its closest paralogue TM9SF2, were both required for bacterial internalisation in S2 cells, where they displayed partial redundancy. Our study highlights the contribution of phagocytes to host defence in an organism possessing a complex innate immune response and suggests an evolutionarily conserved function of TM9SF4 in eukaryotic phagocytes.