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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 Biossensoriais/métodos , Fibras Ópticas , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos/análise , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Ouro/química , Limite de Detecção , Ratos , Ressonância de Plasmônio de Superfície/instrumentação , Propriedades de SuperfícieRESUMO
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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Molecular spin crossover complexes are promising candidates for mechanical actuation purposes. The relationships between their crystal structure and mechanical properties remain, however, not well understood. In this study, combining high pressure synchrotron X-ray diffraction, nuclear inelastic scattering, and micromechanical measurements, we assessed the effective macroscopic bulk modulus ( B = 11.5 ± 1.5 GPa), Young's modulus ( Y = 10.9 ± 1.0 GPa), and Poisson's ratio (ν = 0.34 ± 0.04) of the spin crossover complex [FeII(HB(tz)3)2] (tz = 1,2,4-triazol-1-yl). Crystal structure analysis revealed a pronounced anisotropy of the lattice compressibility, which was correlated with the difference in spacing between the molecules as well as by the distribution of the stiffest C-H···N interactions in different crystallographic directions. Switching the molecules from the low spin to the high spin state leads to a remarkable drop of the Young's modulus to 7.1 ± 0.5 GPa both in bulk and thin film samples. The results highlight the application potential of these films in terms of strain (ε = -0.17 ± 0.05%), recoverable stress (σ = -21 ± 1 MPa), and work density ( W/V = 15 ± 6 mJ/cm3).
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We investigate the pressure-driven transport of particles 200 or 300 nm in diameter in shallow microfluidic channels â¼1 µm in height with a bottom wall characterized by a high roughness amplitude of â¼100 nm. This study starts with the description of an assay to generate cracks in hydrophilic thin polymer films together with a structural characterization of these corrugations. Microfluidic chips of variable height are then assembled on top of these rough surfaces, and the transport of particles is assessed by measuring the velocity distribution function for a set of pressure drops. We specifically detect anomalous transport properties for rough surfaces. The maximum particle velocity at the centerline of the channel is comparable to that obtained with smooth surfaces, but the average particle velocity increases nonlinearly with the flow rate. We suggest that the change in the boundary condition at the rough wall is not sufficient to account for our data and that the occurrence of contacts between the particle and the surface transports the particle away from the wall and speeds up its motion. We finally draw perspectives for the separation by field-flow fractionation.
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We report on a bistable MEMS device actuated by spin-crossover molecules. The device consists of a freestanding silicon microcantilever with an integrated piezoresistive detection system, which was coated with a 140â nm thick film of the [Fe(HB(tz)3 )2 ] (tz=1,2,4-triazol-1-yl) molecular spin-crossover complex. Switching from the low-spin to the high-spin state of the ferrous ions at 338â K led to a reversible upward bending of the cantilever in agreement with the change in the lattice parameters of the complex. The strong mechanical coupling was also evidenced by the decrease of approximately 66â Hz in the resonance frequency in the high-spin state as well as by the drop in the quality factor around the spin transition.
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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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(1) Backround: Technological advances should foster gains in physicians' efficiency. For example, a reduction of the medical decision time can be enabled by faster biological tests. The main objective of this study was to collect responses from an international panel of physicians on their needs for biomarkers and also to convey the improvement in the outcome to be made possible by the potential development of fast diagnostic tests for these biomarkers. (2) Methods: we distributed a questionnaire on the Internet to physicians. (3) Results: 508 physicians participated in this survey. The mean age was 38 years. General practice and emergency medicine were heavily represented, with 95% CIs of 44% (39.78, 48.41) and 32% (27.84, 35.94)), respectively. The two most represented countries were France (95% CI: 74% (70.20, 77.83)) and the USA (95% CI: 11% (8.65, 14.18)). Ninety-eight percentages of the physicians thought that obtaining cited biomarkers more quickly would be beneficial to their practice and to patient's care. The main biomarkers of interest identified by our panel were troponin (95% CI: 51% (46.24, 54.94)), C-reactive protein (95% CI: 42% (38.03, 46.62)), D-dimer (95% CI: 29% (24.80, 32.68)), and brain natriuretic peptide (95% CI: 13% (10.25, 16.13)). (4) Conclusions: Our study highlights the real technological need for fast biomarker results, which could be provided by biosensors. The relevance of some answers such as troponin is questionable.
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We have previously surveyed a panel of 508 physicians from around the world about which biomarkers would be relevant if obtained in a very short time frame, corresponding to emergency situations (life-threatening or not). The biomarkers that emerged from this study were markers of cardiovascular disease: troponin, D-dimers, and brain natriuretic peptide (BNP). Cardiovascular disease is a group of disorders affecting the heart and blood vessels. At the intersection of medicine, basic research and engineering, biosensors that address the need for rapid biological analysis could find a place of choice in the hospital or primary care ecosystem. Rapid, reliable, and inexpensive analysis with a multi-marker approach, including machine learning analysis for patient risk analysis, could meet the demand of medical teams. The objective of this opinion review, proposed by a multidisciplinary team of experts (physicians, biologists, market access experts, and engineers), is to present cases where a rapid biological response is indeed valuable, to provide a short overview of current biosensor technologies for cardiac biomarkers designed for a short result time, and to discuss existing market access issues.
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In this article, we propose a novel dielectrophoretic continuous flow sorter using planar micro electrodes coupled to a channel constriction. This design enables a high particle sorting efficiency at low voltages while relying on a simple fabrication and integration process. We have numerically simulated the AC electrokinetic effects and the fluid behavior to predict particle trajectories. Simulation results are in accordance with experimental data: 10 and 5 µm polystyrene beads were continuously sorted with <2% errors at flow speeds of 100 µm/s. We were also able to change the particle buffer while sorting beads. Finally, to demonstrate the interest of our device for cell sorting, we also sorted dead and living yeast cells according to their different dielectric properties. Living cell concentration was enriched by a factor of 4 versus dead cell concentration after passing the sorting device.
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Separação Celular/instrumentação , Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Microesferas , Separação Celular/métodos , Simulação por Computador , Eletroforese/métodos , Desenho de Equipamento , Análise de Elementos Finitos , Microeletrodos , Técnicas Analíticas Microfluídicas/métodos , Modelos Teóricos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologiaRESUMO
We present a fluorimetry-based technology for micro-RNA-21 (miR-21) sensing based on the concentration of miR-molecular beacon (MB) complexes and flushing of unbound MB. This concentration module consists of a microfluidic channel with the shape of a funnel operated with electrohydrodynamic actuation. We report a limit of detection of 2 pM in less than 1 min for miR-21 alone, and then demonstrate that miR-21 levels, measured in fine needle biopsy samples, from patients with pancreatic cancer correlate with the reference technique of reverse-transcription polymerase chain reaction (RT-PCR). Altogether, this technology has promising clinical performances for the follow-up of patients with cancer.
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Técnicas Biossensoriais , MicroRNAs , Humanos , MicroRNAs/análise , Microfluídica , RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The fabrication and integration of sub-millimeter magnetic materials into predefined circuits is of major importance for the realization of portable devices designed for telecommunications, automotive, biomedical, and space applications but remains highly challenging. We report here a versatile approach for the fabrication and direct integration of nanostructured magnetic materials of controlled shaped at specific locations onto silicon substrates. The magnetophoresis-assisted capillary assembly of magnetic nanoparticles, either spherical or anisotropic, leads to the fabrication of high-performance Co-based permanent magnets and Fe-based supercrystals. Integrated sub-millimeter magnets as well as millimeter self-standing magnets exhibiting magnetic properties competing with NdFeB-based composites were obtained through this cost- and time-efficient process. The proof-of-concept of electromagnetic actuation of a micro-electromechanical system cantilever by means of these supercrystals highlights their potentiality as efficient integrated magnetic materials within nomadic devices.
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The acceleration of climatic, digital, and health challenges is testing scientific communities. Scientists must provide concrete answers in terms of technological solutions to a society which expects immediate returns on the public investment. We are living such a scenario on a global scale with the pandemic crisis of COVID-19 where expectations for virological and serological diagnosis tests have been and are still gigantic. In this Perspective, we focus on a class of biosensors (mechanical biosensors) which are ubiquitous in the literature in the form of high performance, sensitive, selective, low-cost biological analysis systems. The spectacular development announced in their performance in the last 20 years suggested the possibility of finding these mechanical sensors on the front line of COVID-19, but the reality was quite different. We analyze the cause of this rendez-vous manqué, the operational criteria that kept these biosensors away from the field, and we indicate the pitfalls to avoid in the future in the development of all types of biosensors of which the ultimate goal is to be immediately operational for the intended application.
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Técnicas Biossensoriais/métodos , COVID-19/diagnóstico , Sistemas Microeletromecânicos , COVID-19/virologia , Humanos , Limite de Detecção , Prognóstico , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Selective Area van der Waals Epitaxy (SAVWE) of III-Nitride device has been proposed recently by our group as an enabling solution for h-BN-based device transfer. By using a patterned dielectric mask with openings slightly larger than device sizes, pick-and-place of discrete LEDs onto flexible substrates was achieved. A more detailed study is needed to understand the effect of this selective area growth on material quality, device performance and device transfer. Here we present a study performed on two types of LEDs (those grown on h-BN on patterned and unpatterned sapphire) from the epitaxial growth to device performance and thermal dissipation measurements before and after transfer. Millimeter-size LEDs were transferred to aluminum tape and to silicon substrates by van der Waals liquid capillary bonding. It is shown that patterned samples lead to a better material quality as well as improved electrical and optical device performances. In addition, patterned structures allowed for a much better transfer yield to silicon substrates than unpatterned structures. We demonstrate that SAVWE, combined with either transfer processes to soft or rigid substrates, offers an efficient, robust and low-cost heterogenous integration capability of large-size devices to silicon for photonic and electronic applications.
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In the era of precision medicine, the success of clinical trials, notably for patients diagnosed with cancer, strongly relies on biomarkers with pristine clinical value but also on robust and versatile analytical technologies to ensure proper patients' stratification and treatment. In this review, we will first address whether plasmatic and salivary microRNAs can be considered as a reliable source of biomarkers for cancer diagnosis and prognosis. We will then discuss the pre-analytical steps preceding miRNA quantification (from isolation to purification), and how such process could be biased and time-consuming. Next, we will review the most recent tools derived from micro- and nano-technologies for microRNA detection available to date and how they may compete with current standards. This review will prioritize publications using relevant biological samples. The significance of various physical transduction schemes (mechanical, optical, electrical, etc.) for biological detection will be compared, and pros and cons of each method will be widely discussed. Finally, we will debate on how micro and nanotechnologies could widespread the use of biomarkers in modern medicine, to help manage patients with serious diseases such as cancer.
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The development of advanced techniques of fabrication of three-dimensional (3D) microenvironments for the study of cell growth and proliferation has become one of the major motivations of material scientists and bioengineers in the past decade. Here, we present a novel residueless 3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS emulsion casting and subsequent curing process in temperature-/pressure-controlled environment. Scanning electron microscopy and X-ray microcomputed tomography allowed us to investigate the impact of those parameters on the microarchitecture of the porous structure. We demonstrated that the optimized emulsion casting process gives rise to large-scale and highly interconnected network with pore size ranging from 500 µm to 1.5 mm that turned out to be nicely adapted to 3D cell culture. Experimental cell culture validations were performed using SaOS-2 (osteosarcoma) cell lines. Epifluorescence and deep penetration imaging techniques as two-photon confocal microscopy unveiled information about cell morphology and confirmed a homogeneous cell proliferation and spatial distribution in the 3D porous structure within an available volume larger than 1 cm3. These results open alternative scenarios for the fabrication and integration of porous scaffolds for the development of 3D cell culture platforms.
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Microambiente Celular , Teste de Materiais , Silicones/química , Linhagem Celular Tumoral , Emulsões , Humanos , Porosidade , Alicerces Teciduais , ÁguaRESUMO
We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.
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Testes Diagnósticos de Rotina/métodos , Doenças do Sistema Nervoso/diagnóstico , Doenças Neuromusculares/diagnóstico , Expansão das Repetições de Trinucleotídeos , Humanos , Dispositivos Lab-On-A-Chip , Doenças do Sistema Nervoso/patologia , Doenças Neuromusculares/patologia , Sensibilidade e EspecificidadeRESUMO
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 Biossensoriais , Silício/química , Impedância Elétrica , Técnicas Eletroquímicas , Membranas Artificiais , NanoporosRESUMO
In this work, we demonstrate that the analysis of spatially resolved nanofluidic-embedded biosensors permits the fast and direct discrimination of single-nucleotide difference (SND) within oligonucleotide sequences in a single step interaction. We design a sensor with a linear dimension much larger than the channel depth in order to ensure that the reaction over the whole sensor is limited by the convection rate. Thus, the targets are fully collected, inducing a nonuniform spatial hybridization profile. We also use the nanoscale height of the channel, which enables us to minimize the amount of labeled molecules flowing over the sensor and hence to reduce the fluorescence background, to carry out real-time hybridization detection by fluorescence microscopy. Taken together, these design rules allow us to show that the spatial hybridization profile depends on the duplex affinity, and we speculate that the on and off-rate constants can be inferred during target injection, which is not possible in local analysis where the dissociation step through rinsing must be conducted. We finally manage to discriminate a GT mismatch on a microRNA sequence by optimizing the interaction temperature and the probe design after a few minutes of interaction in a single step protocol. This work may be applied to any biosensing transduction scheme with spatial resolution, e.g., surface plasmon resonance imaging, integrated into nanofluidic channels for applications where high oligonucleotide sequence selectivity and short analysis times are required.
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Técnicas Biossensoriais/métodos , Técnicas Analíticas Microfluídicas , Nanotecnologia , Nucleotídeos/análise , Nucleotídeos/química , Oligonucleotídeos/química , Microscopia de Fluorescência , Hibridização de Ácido NucleicoRESUMO
Kinetic monitoring of protein interactions offers insights to their corresponding functions in cellular processes. Surface plasmon resonance (SPR) is the current standard tool used for label-free kinetic assays; however, costly and sophisticated setups are required, decreasing its accessibility to research laboratories. We present a cost-effective nanofluidic-based immunosensor for low-noise real-time kinetic measurement of fluorescent-labeled protein binding. With the combination of fluorescence microscopy and reversed buffer flow operation, association and dissociation kinetics can be accessed in one single experiment without extra buffer loading step, which results in a simplified operation and reduced time of analysis compared to typical microfluidic immunoassays. Kinetic constants of two representative protein-ligand binding pairs (streptavidin/biotin; IgG/anti-IgG) were quantified. The good agreement of extracted rate constants with literature values and analogous SPR measurements indicates that this approach is applicable to study protein interactions of medium- and high-affinities with a limit of detection down to 1 pM, regardless of the analyte size.
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Anticorpos Imobilizados/química , Imunoglobulina G/análise , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Animais , Biotina/química , Desenho de Equipamento , Imunoensaio/instrumentação , Cinética , Camundongos , Ligação Proteica , Estreptavidina/químicaRESUMO
DNA size separation followed by purification and enrichment constitute essential operations for genetic engineering. These processes are mostly carried out using DNA electrophoresis in gels or in polymer solutions, a well-established yet lengthy technique which has been notably improved using Lab-on-Chip technologies. So far, innovations for DNA separation or enrichment have been mostly undertaken separately, and we present an approach that allows us to perform these two processes simultaneously for DNA fragments spanning 0.2-50 kilo base pairs (kbp) in length. Our technology involves an electric field and a counter hydrodynamic flow in viscoelastic liquids, in which we show the occurrence of transverse forces oriented toward the walls. These forces increase with DNA molecular weight (MW) and hence induce a progressive reduction in DNA migration speed that triggers size separation in microfluidic channels as well as in capillaries. The separation of MW markers in the range 1-50 kbp is achieved in 15 minutes, thus outperforming gel electrophoresis that takes â¼3 hours for this sample. Furthermore, the use of a funnel, where electric and flow fields are modulated spatially, enables us to adjust the transverse forces so as to stall the motion of DNA molecules at a position where they accumulate at factors of up to 1000 per minute. In this configuration, we establish that the operations of DNA enrichment and separation can be carried out simultaneously for the bands of a DNA MW marker between 0.2-1.5 kbp diluted at 0.02 ng µL(-1) in 30 s. Altogether, our technology, which can readily be integrated as an in-line module in Lab-on-Chips, offers unique opportunities for sample preparation and analysis of minute genomic samples.