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This work depicts the original combination of electrochemiluminescence (ECL) and bipolar electrochemistry (BPE) to map in real-time the oxidation of silicon in microchannels. We fabricated model silicon-PDMS microfluidic chips, optionally containing a restriction, and monitored the evolution of the surface reactivity using ECL. BPE was used to remotely promote ECL at the silicon surface inside microfluidic channels. The effects of the fluidic design, the applied potential and the resistance of the channel (controlled by the fluidic configuration) on the silicon polarization and oxide formation were investigated. A potential difference down to 6â V was sufficient to induce ECL, which is two orders of magnitude less than in classical BPE configurations. Increasing the resistance of the channel led to an increase in the current passing through the silicon and boosted the intensity of ECL signals. Finally, the possibility of achieving electrochemical reactions at predetermined locations on the microfluidic chip was investigated using a patterning of the silicon oxide surface by etched micrometric squares. This ECL imaging approach opens exciting perspectives for the precise understanding and implementation of electrochemical functionalization on passivating materials. In addition, it may help the development and the design of fully integrated microfluidic biochips paving the way for development of original bioanalytical applications.
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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.
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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.
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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
Bipolar electrochemistry (BPE) is a powerful method based on the wireless polarization of a conductive object that induces the asymmetric electroactivity at its two extremities. A key physical limitation of BPE is the size of the conductive object because the shorter the object, the larger is the potential necessary for sufficient polarization. Micrometric and nanometric objects are thus extremely difficult to address by BPE due to the very high potentials required, in the order of tens of kV or more. Herein, the synergetic actions of BPE and of planar micropores integrated in a microfluidic device lead to the spatial confinement of the potential drop at the level of the solid-state micropore, and thus to a locally enhanced polarization of a bipolar electrode. Electrochemiluminescence (ECL) is emitted in half of the electroactive micropore and reveals the asymmetric polarization in this spatial restriction. Micrometric deoxidized silicon electrodes located in the micropore are polarized at a very low potential (7 V), which is more than 2 orders of magnitude lower compared to the classic bipolar configurations. This behavior is intrinsically associated with the unique properties of the micropores, where the sharp potential drop is focused. The presented approach offers exciting perspectives for BPE of micro/nano-objects, such as dynamic BPE with objects passing through the pores or wireless ECL-emitting micropores.
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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
Protein surface patterning is employed in a broad spectrum of applications ranging from protein microarray analysis to 2D cell organization. However, limitations arise because of the highly sensitive nature of proteins requiring careful handling to ensure their structural and functional integrity during the grafting process. Here, we describe a patterning protocol that keeps proteins in an aqueous environment during their immobilization, avoiding the loss of their biological activity. The procedure is based on the UV-mediated removal of polyethylene glycol self-assembled monolayers in a transparent microfluidic chamber, giving access to micrometric motifs of predefined geometries. Afterward, modified proteins can be grafted on the photopatterned domains. We also studied the influence of reactive oxygen species for a better understanding of the chemical mechanism involved in this process. Finally, as a proof of concept, a protein microarray was created with this process using cell-capturing antibodies to immobilize human blood cells, confirming the functionality of the arrayed proteins.
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Proteínas/química , Humanos , Microfluídica , Polietilenglicoles , Análisis por Matrices de Proteínas , Propiedades de Superficie , AguaRESUMEN
Several optical surface sensing techniques, such as Surface Plasmon Resonance (SPR), work by imaging the base of a prism by one of its faces. However, such a fundamental optical concern has not been fully analyzed and understood so far, and spatial resolution remains a critical and controversial issue. In SPR, the propagation length L(x) of the surface plasmon waves has been considered as the limiting factor. Here, we demonstrate that for unoptimized systems geometrical aberrations caused by the prism can be more limiting than the propagation length. By combining line-scan imaging mode with optimized prisms, we access the ultimate lateral resolution which is diffraction-limited by the object light diffusion. We describe several optimized configurations in water and discuss the trade-off between L(x) and sensitivity. The improvement of resolution is confirmed by imaging micro-structured PDMS stamps and individual living eukaryote cells and bacteria on field-of-view from 0.1 to 20 mm(2).
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Microscopía/instrumentación , Resonancia por Plasmón de Superficie/instrumentación , Diseño de EquipoRESUMEN
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.
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The use of biological-probe-modified solid-state pores in biosensing is currently hindered by difficulties in pore-wall functionalization. The surface to be functionalized is small and difficult to target and is usually chemically similar to the bulk membrane. Herein, we demonstrate the contactless electrofunctionalization (CLEF) approach and its mechanism. This technique enables the one-step local functionalization of the single pore wall fabricated in a silica-covered silicon membrane. CLEF is induced by polarization of the pore membrane in an electric field and requires a sandwich-like composition and a conducting or semiconducting core for the pore membrane. The defects in the silica layer of the micropore wall enable the creation of an electric pathway through the silica layer, which allows electrochemical reactions to take place locally on the pore wall. The pore diameter is not a limiting factor for local wall modification using CLEF. Nanopores with a diameter of 200 nm fabricated in a silicon membrane and covered with native silica layer have been successfully functionalized with this method, and localized pore-wall modification was obtained. Furthermore, through proof-of-concept experiments using ODN-modified nanopores, we show that functionalized nanopores are suitable for translocation-based biosensing.
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Técnicas Biosensibles/métodos , Microtecnología/métodos , Nanoporos , Electricidad , Membranas Artificiales , Dióxido de Silicio/químicaRESUMEN
The analysis of volatile organic compounds (VOCs) is an important issue in various domains. For this, electronic noses (eN) are very promising as novel analytical tools that are portable, inexpensive, and efficient for reliable and rapid analyses. Recently, we have demonstrated that surface plasmon resonance imaging (SPRI) is especially interesting for the development of eNs dedicated for gas-phase analysis of VOCs. To further improve the performance of the eN based on SPRI, in this study, we investigated the influence of the LED wavelength on the sensitivity of the system. For this, a complete theoretical study together with a related experimental investigation for the validation were carried out. We have shown that the wavelength of the light source has an impact on the surface sensitivity of SPRI for the detection of VOCs. Indeed, in the studied wavelength range from 530 nm to 740 nm, both bulk sensitivity and surface sensitivity increase as the wavelength increases with good coherence between theoretical and experimental results. With the optimal LED wavelength, the detection limits of our eN reach low ppb range for VOC such as 1-butanol.
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Foodborne pathogens are of significant concern in the agrifood industry and the development of associated rapid detection and identification methods are of major importance. This paper describes the novel use of resolution-optimized prism-based surface plasmon resonance imaging (RO-SPRI) and data processing for the detection of the foodborne pathogens Listeria monocytogenes and Listeria innocua. With an imaging spatial resolution on the order of individual bacteria (2.7 ± 0.5 µm × 7.9 ± 0.6 µm) over a field of view 1.5 mm2, the RO-SPRI system enabled accurate counting of individual bacteria on the sensor surface. Using this system, we demonstrate the detection of two species of Listeria at an initial concentration of 2 × 102 CFU mL-1 in less than 7 hours. The surface density of bacteria at the point of positive detection was 15 ± 4 bacteria per mm2. Our approach offers great potential for the development of fast specific detection systems based on affinity monitoring.
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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.
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Técnicas Biosensibles , Silicio/química , Impedancia Eléctrica , Técnicas Electroquímicas , Membranas Artificiales , NanoporosRESUMEN
The adult vasculature is comprised of three distinct compartments: the arteries, which carry blood away from the heart and display a divergent flow pattern; the capillaries, where oxygen and nutrient delivery from blood to tissues, as well as metabolic waste removal, occurs; and the veins, which carry blood back to the heart and are characterized by a convergent flow pattern. These compartments are organized in series as regard to flow, which proceeds from the upstream arteries to the downstream veins through the capillaries. However, the spatial organization is more complex, as veins may often be found paralleling the arteries. The factors that control the morphogenesis of this hierarchically branched vascular network are not well characterized. Here, we explain how arteries exert a morphological control on the venous pattern. Indeed, during vertebrate development, the following transition may be observed in the spatial organization of the vascular system: veins first develop in series with the arteries, the arterial and venous territories being clearly distinct in space (cis-cis configuration). But after some time, new veins grow parallel to the existing arteries, and the arterial and venous territories become overlapped, with extensive and complex intercalation and interdigitation. Using physical arguments, backed up by experimental evidence (biological data from the literature and in situ optical and mechanical measurements of the chick embryo yolk-sac and midbrain developing vasculatures), we explain how such a transition is possible and why it may be expected with generality, as organisms grow. The origin of this transition lies in the remodeling of the capillary tissue in the vicinity of the growing arteries. This remodeling lays down a prepattern for further venous growth, parallel to the existing arterial pattern. Accounting for the influence of tissue growth, we show that this prepatterned path becomes favored as the body extends. As a consequence, a second flow route with veins paralleling the arteries (cis-trans configuration) emerges when the tissue extends. Between the cis-cis and cis-trans configurations, all configurations are in principle possible, and self-organization of the vessels contributes to determining their exact pattern. However, the global aspect depends on the size at which the growth stops and on the growth rate.
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Arterias/embriología , Arterias/crecimiento & desarrollo , Modelos Biológicos , Morfogénesis/fisiología , Venas/embriología , Venas/crecimiento & desarrollo , Animales , Embrión de Pollo , Simulación por Computador , Retroalimentación/fisiologíaRESUMEN
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).
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Anticuerpos Inmovilizados/química , Interferón gamma/análisis , Interleucina-2/análisis , Dispositivos Laboratorio en un Chip , Resonancia por Plasmón de Superficie/instrumentación , Linfocitos T/inmunología , Adulto , Anticuerpos Inmovilizados/inmunología , Supervivencia Celular , Diseño de Equipo , Humanos , Interferón gamma/inmunología , Interleucina-2/inmunología , Análisis por Matrices de Proteínas/instrumentación , Linfocitos T/químicaRESUMEN
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
BACKGROUND: Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. METHODOLOGY/PRINCIPAL FINDINGS: We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. CONCLUSIONS/SIGNIFICANCE: The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures.
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Linfocitos B/citología , Separación Celular/métodos , Bazo/citología , Linfocitos T/citología , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Separación Celular/instrumentación , Filtración/instrumentación , Filtración/métodos , Inmunoconjugados/química , Ratones , Microesferas , Oligodesoxirribonucleótidos/química , Polímeros/química , Pirroles/química , Dióxido de Silicio/químicaRESUMEN
Many biological samples are composed of several cell types. Qualitative and quantitative analysis of these complex mixtures is of major interest for both diagnostic and biomedical applications. Because large amounts of biological material are often challenging to collect, tremendous efforts have been made for a decade to design miniaturized platforms-such as lab-on-a-chip or microarrays-to run sensitive and reliable analysis from tiny quantities of starting material. Although barely explored so far, the release of resolved cellular samples constitutes an exciting strategy for further cell analysis. Herein, we propose a DNA-based biochip suitable for cell-type analysis in a label-free manner. The DNA-array is firstly converted into antibody-array using antibody-DNA conjugates. These protein-DNA hybrid molecules are chemically synthesized by covalent coupling of short oligonucleotides to antibodies directed against cell-type specific markers. We show not only specific capture of primary spleen cells on protein-DNA microarray spots but also their fast and specific orthogonal release according to the antibody-DNA combinations by incorporating restriction sites in DNA. Both molecular and cellular interactions occurring on the biochip are monitored by surface plasmon resonance (SPR) imaging. This optical technique turns out to be a powerful way to monitor, in real-time, biological interactions occurring on the microarrayed features.
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Anticuerpos/química , ADN/química , Linfocitos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Resonancia por Plasmón de Superficie/métodos , Animales , Comunicación Celular , Mezclas Complejas/química , Ratones , Ratones Endogámicos C57BL , Bazo/inmunologíaRESUMEN
It is getting increasingly evident that physical properties such as elastoviscoplastic properties of living materials are quite important for the process of tissue development, including regulation of genetic pathways. Measuring such properties in vivo is a complicated and challenging task. In this paper, we present an instrument, a scanning air puff tonometer, which is able to map point by point the viscoelastic properties of flat or gently curved soft materials. This instrument is an improved version of the air puff tonometer used by optometrists, with important modifications. The instrument allows one to obtain a direct insight into gradients of material properties in vivo. The instrument capabilities are demonstrated on substances with known elastoviscoplastic properties and several biological objects. On the basis of the results obtained, the role of the gradients of elastoviscoplastic properties is outlined for the process of angiogenesis, limb development, bacterial colonies expansion, etc. which is important for bridging the gaps in the theory of the tissue development and highlighting new possibilities for tissue engineering, based on a clarification of the role of physical features in developing biological material.