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Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as â¼1 pg (â¼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.
Assuntos
Técnicas Biossensoriais , DNA Circular/análise , Resistência Microbiana a Medicamentos/genética , Dispositivos Lab-On-A-Chip , Microscopia de Fluorescência/métodos , Reação em Cadeia da Polimerase/métodos , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Carbocianinas/química , Sondas de DNA/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , Dimetilpolisiloxanos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Expressão Gênica , Humanos , Meticilina/farmacologia , Microscopia de Fluorescência/economia , Microscopia de Fluorescência/instrumentação , Mutação , Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/instrumentação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mitoribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.
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Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Rotenona/análogos & derivados , Animais , Anti-Helmínticos/farmacologia , Caenorhabditis elegans/genética , Resistência a Medicamentos/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Rotenona/farmacologiaRESUMO
We detect by optical microscopy Au and fluorescent nanoparticles (NPs) during their motion in water-based medium, using an array of dielectric microspheres that are patterned in a microwell array template. The microspheres act as lenses focusing the light originating from a microscope objective into so-called photonic nanojets that expose the medium within a microfluidic channel. When a NP is randomly transported through a nanojet, its backscattered light (for a bare Au NP) or its fluorescent emission is instantaneously detected by video microscopy. Au NPs down to 50 nm in size, as well as fluorescent NPs down to 20 nm in size, are observed by using a low magnification/low numerical aperture microscope objective in bright-field or fluorescence mode, respectively. Compared to the NPs present outside of the photonic nanojets, the light scattering or fluorescence intensity of the NPs in the nanojets is typically enhanced by up to a factor of â¼40. The experimental intensity is found to be proportional to the area occupied by the NP in the nanojet. The technique is also used for immunodetection of biomolecules immobilized on Au NPs in buffer and, in future, it may develop into a versatile tool to detect nanometric objects of environmental or biological importance, such as NPs, viruses, or other biological agents.
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We describe the concept of magnetic particle-scanning for on-chip detection of biomolecules: a magnetic particle, carrying a low number of antigens (Ag's) (down to a single molecule), is transported by hydrodynamic forces and is subjected to successive stochastic reorientations in an engineered magnetic energy landscape. The latter consists of a pattern of substrate-bound small magnetic particles that are functionalized with antibodies (Ab's). Subsequationuent counting of the captured Ag-carrying particles provides the detection signal. The magnetic particle-scanning principle is investigated in a custom-built magneto-microfluidic chip and theoretically described by a random walk-based model, in which the trajectory of the contact point between an Ag-carrying particle and the small magnetic particle pattern is described by stochastic moves over the surface of the mobile particle, until this point coincides with the position of an Ag, resulting in the binding of the particle. This model explains the particular behavior of previously reported experimental dose-response curves obtained for two different ligand-receptor systems (biotin/streptavidin and TNF-α) over a wide range of concentrations. Our model shows that magnetic particle-scanning results in a very high probability of immunocomplex formation for very low Ag concentrations, leading to an extremely low limit of detection, down to the single molecule-per-particle level. When compared to other types of magnetic particle-based surface coverage assays, our strategy was found to offer a wider dynamic range (>8 orders of magnitude), as the system does not saturate for concentrations as high as 10(11) Ag molecules in a 5 µL drop. Furthermore, by emphasizing the importance of maximizing the encounter probability between the Ag and the Ab to improve sensitivity, our model also contributes to explaining the behavior of other particle-based heterogeneous immunoassays.
Assuntos
Anticorpos Imobilizados/química , Imunoensaio/instrumentação , Magnetismo/instrumentação , Imãs/química , Técnicas Analíticas Microfluídicas/instrumentação , Prata/química , Animais , Biotina/química , Bovinos , Desenho de Equipamento , Fenômenos Magnéticos , Camundongos , Modelos Químicos , Estreptavidina/análise , Fator de Necrose Tumoral alfa/sangueRESUMO
Lifespan is influenced by complex interactions between genetic and environmental factors. Studying those factors in model organisms of a single genetic background limits their translational value for humans. Here, we mapped lifespan determinants in 85 genetically diverse C. elegans recombinant intercross advanced inbred lines (RIAILs). We assessed molecular profiles - transcriptome, proteome, and lipidome - and life-history traits, including lifespan, development, growth dynamics, and reproduction. RIAILs exhibited large variations in lifespan, which positively correlated with developmental time. Among the top candidates obtained from multi-omics data integration and QTL mapping, we validated known and novel longevity modulators, including rict-1, gfm-1 and mltn-1. We translated their relevance to humans using UK Biobank data and showed that variants in RICTOR and GFM1 are associated with an elevated risk of age-related heart disease, dementia, diabetes, kidney, and liver diseases. We organized our dataset as a resource (https://lisp-lms.shinyapps.io/RIAILs/) that allows interactive explorations for new longevity targets.
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We have demonstrated hyperspectral tip-enhanced Raman imaging on dielectric substrates using linearly polarized light and nanofabricated coaxial antenna tips. A full Raman spectrum was acquired at each pixel of a 256 by 256 pixel contact-mode atomic force microscope image of carbon nanotubes grown on a fused silica microscope coverslip, allowing D and G mode intensity and D-mode peak shifts to be measured with â¼20 nm spatial resolution. Tip enhancement was sufficient to acquire useful Raman spectra in 50-100 ms. Coaxial scan probes combine the efficiency and enhanced, ultralocalized optical fields of plasmonically coupled antennae with the superior topographical imaging properties of sharp metal tips. The yield of the coaxial tip fabrication process is close to 100%, and the tips are sufficiently durable to support hours of contact-mode force microscope imaging. Our coaxial probes avoid the limitations associated with the "gap-mode" imaging geometry used in most tip-enhanced Raman studies to date, where a sharp metal tip is held â¼1 nm above a metallic substrate with the sample located in the gap.
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Mitochondrial respiration is a key signature for the assessment of mitochondrial functioning and mitochondrial dysfunction is related to many diseases including metabolic syndrome and aging-associated conditions. Here, we present a microfluidic Caenorhabditis elegans culture system with integrated luminescence-based oxygen sensing. The material used for the fabrication of the microfluidic chip is off-stoichiometry dual-cure thiol-ene-epoxy (OSTE+), which is well-suited for reliably recording on-chip oxygen consumption rates (OCR) due to its low gas permeability. With our microfluidic approach, it was possible to confine a single nematode in a culture chamber, starting from the L4 stage and studying it over a time span of up to 6 days. An automated protocol for successive worm feeding and OCR measurements during worm development was applied. We found an increase of OCR values from the L4 larval stage to adulthood, and a continuous decrease as the worm further ages. In addition, we performed a C. elegans metabolic assay in which exposure to the mitochondrial uncoupling agent FCCP increased the OCR by a factor of about two compared to basal respiration rates. Subsequent treatment with sodium azide inhibited completely mitochondrial respiration.
Assuntos
Dispositivos Lab-On-A-Chip , Oxigênio/análise , Animais , Caenorhabditis elegans/metabolismo , Oxigênio/metabolismo , Consumo de OxigênioRESUMO
The nematode Caenorhabditis elegans has been extensively used as a model multicellular organism to study the influence of osmotic stress conditions and the toxicity of chemical compounds on developmental and motility-associated phenotypes. However, the several-day culture of nematodes needed for such studies has caused researchers to explore alternatives. In particular, C. elegans embryos, due to their shorter developmental time and immobile nature, could be exploited for this purpose, although usually their harvesting and handling is tedious. Here, we present a multiplexed, high-throughput and automated embryo phenotyping microfluidic approach to observe C. elegans embryogenesis after the application of different chemical compounds. After performing experiments with up to 800 embryos per chip and up to 12 h of time-lapsed imaging per embryo, the individual phenotypic developmental data were collected and analyzed through machine learning and image processing approaches. Our proof-of-concept platform indicates developmental lag and the induction of mitochondrial stress in embryos exposed to high doses (200 mM) of glucose and NaCl, while small doses of sucrose and glucose were shown to accelerate development. Overall, our new technique has potential for use in large-scale developmental biology studies and opens new avenues for very rapid high-throughput and high-content screening using C. elegans embryos.
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Caenorhabditis elegans (C. elegans) constitutes an important model organism for use in nutrition and aging studies. We report a novel method for studying the dynamics of Escherichia coli (E. coli) bacterial transit through the worms' intestine. A microfluidic chip was designed for alternating C. elegans on-chip culture and immobilization, thereby enabling periodic high-resolution time-lapse imaging at single-worm resolution over several days. Immobilization was achieved in a reversible way using arrays of tapered channels suitable for assay parallelization. Dedicated C. elegans feeding protocols were applied. Two E. coli bacterial strains, HT115 and OP50, respectively labeled with green fluorescent protein (GFP) and red fluorescent protein (RFP), were used as food source and imaged with fluorescence microscopy techniques to measure relevant parameters of the bacterial transit process. Feeding behavior and E. coli transit dynamics in the whole intestinal tract of the worms were characterized in an automated way over the first 3 days of adulthood, revealing both fast transit phenomena and variations in microbial accumulation. In particular, we studied the bacterial food transit periodicity in wild-type and eat-2 (ad465) mutant C. elegans strains in both trapped and free-swimming conditions. In order to further demonstrate the versatility of our microfluidic platform, we also studied drug-induced modifications of the bacterial transit by measuring the response of the worms' intestine to exposure to the neurotransmitter serotonin.
Assuntos
Caenorhabditis elegans , Microfluídica , Nematoides , Animais , Bactérias , Escherichia coli/genéticaRESUMO
The nematode Caenorhabditis elegans is increasingly used as a model for human biology. However, in vivo culturing platforms for C. elegans allowing high-content phenotyping during their life cycle in an automated fashion are lacking so far. Here, a multiplexed microfluidic platform for the rapid high-content phenotyping of populations of C. elegans down to single animal resolution is presented. Nematodes are (i) reversibly and regularly confined during their life inside tapered channels for imaging fluorescence signal expression and to measure their growth parameters, and (ii) allowed to freely move in microfluidic chambers, during which the swimming behavior was video-recorded. The obtained data sets are analyzed in an automated way and 19 phenotypic parameters are extracted. Our platform is employed for studying the effect of bacteria dilution, a form of dietary restriction (DR) in nematodes, on a worm model of Huntington's disease and demonstrates the influence of DR on disease regression.
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Caenorhabditis elegans/fisiologia , Larva/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Animais , Técnicas de Cultura/instrumentação , Técnicas de Cultura/métodos , Modelos Animais de Doenças , Desenho de Equipamento , FenótipoRESUMO
A major step for the validation of medical drugs is the screening on whole organisms, which gives the systemic information that is missing when using cellular models. Caenorhabditis elegans is a soil worm that catches the interest of researchers who study systemic physiopathology (e.g., metabolic and neurodegenerative diseases) because: (1) its large genetic homology with humans supports translational analysis; (2) worms are much easier to handle and grow in large amounts compared with rodents, for which (3) the costs and (4) the ethical concerns are substantial. Here, we demonstrate how multimodal optical imaging on such an organism can provide high-content information relevant to the drug development pipeline (e.g., mode-of-action identification, dose-response analysis), especially when combined with on-chip multiplexing capability. After designing a microfluidic array to select small separated populations of C. elegans, we combine fluorescence and bright-field imaging along with high-throughput feature recognition and signal detection to enable the identification of the mode-of-action of an antibiotic. For this purpose, we use a genetically encoded fluorescence reporter of mitochondrial stress, which we studied in living specimens during their entire development. Furthermore, we demonstrate real-time, very large field-of-view capability on multiplexed motility assays for the assessment of the dose-response relation of an anesthetic.
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Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Dispositivos Lab-On-A-Chip , Imagem Multimodal , Animais , Caenorhabditis elegans , Técnicas Analíticas Microfluídicas/métodos , Imagem Óptica/métodosRESUMO
Small molecules inhibitors are powerful tools for studying multiple aspects of cell biology and stand at the forefront of drug discovery pipelines. However, in the early Caenorhabditis elegans (C. elegans) embryo, which is a powerful model system for cell and developmental biology, the use of small molecule inhibitors has been limited by the impermeability of the embryonic eggshell, the low-throughput manual embryo isolation methods, and the lack of well-controlled drug delivery protocols. This work reports a fully integrated microfluidic approach for studies of C. elegans early embryogenesis, including the possibility of testing small molecule inhibitors with increased throughput and versatility. The setup enables robust on-chip extraction of embryos from gravid adult worms in a dedicated pillar array chamber by mechanical compression, followed by rapid fluidic transfer of embryos into an adjacent microtrap array. Parallel analysis of ≈100 embryos by high-resolution time-lapse imaging from the one-cell stage zygote until hatching can be performed with this device. The implementation of versatile microfluidic protocols, in particular time-controlled and reversible drug delivery to on-chip immobilized embryos, demonstrates the potential of the device for biochemical and pharmacological assays.
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The nematode Caenorhabditis elegans is an important model organism for biomedical research and genetic studies relevant to human biology and disease. Such studies are often based on high-resolution imaging of dynamic biological processes in the worm body tissues, requiring well-immobilized and physiologically active animals in order to avoid movement-related artifacts and to obtain meaningful biological information. However, existing immobilization methods employ the application of either anesthetics or servere physical constraints, by using glue or specific microfluidic on-chip mechanical structures, which in some cases may strongly affect physiological processes of the animals. Here, we immobilize C. elegans nematodes by taking advantage of a biocompatible and temperature-responsive hydrogel-microbead matrix. Our gel-based immobilization technique does not require a specific chip design and enables fast and reversible immobilization, thereby allowing successive imaging of the same single worm or of small worm populations at all development stages for several days. We successfully demonstrated the applicability of this method in challenging worm imaging contexts, in particular by applying it for high-resolution confocal imaging of the mitochondrial morphology in worm body wall muscle cells and for the long-term quantification of number and size of specific protein aggregates in different C. elegans neurodegenerative disease models. Our approach was also suitable for immobilizing other small organisms, such as the larvae of the fruit fly Drosophila melanogaster and the unicellular parasite Trypanosoma brucei. We anticipate that this versatile technique will significantly simplify biological assay-based longitudinal studies and long-term observation of small model organisms.
Assuntos
Caenorhabditis elegans/anatomia & histologia , Hidrogéis , Imobilização/métodos , Microesferas , Imagem Óptica/métodos , Animais , Caenorhabditis elegans/ultraestrutura , Mitocôndrias/ultraestrutura , Músculos/diagnóstico por imagem , Músculos/ultraestrutura , Interferência de RNARESUMO
The organism Caenorhabditis elegans is a performant model system for studying human biological processes and diseases, but until now all phenome data are produced as population-averaged read-outs. Monitoring of individual responses to drug treatments would however be more informative. Here, a new strategy to track different phenotypic traits of individual C. elegans nematodes throughout their full life-cycle-i.e., embryonic and post-embryonic development, until adulthood onset, differently from life-span-is presented. In an automated fashion, single worms were synchronized, isolated, and cultured from egg to adulthood in a microfluidic device, where their identity was preserved during their whole development. Several phenotypes were monitored and quantified for each animal, resulting in high-content phenome data. Specifically, the method was validated by analyzing the response of C. elegans to doxycycline, an antibiotic fairly well-known to prolong the development and activate mitochondrial stress-response pathways in different species. Interestingly, the obtained extensive single-worm phenome not only confirmed the dramatic doxycycline effect on the worm developmental delay, but more importantly revealed subtle yet severe treatment-dependent phenotypes that are representative of minority subgroups and would have otherwise stayed hidden in an averaged dataset. Such heterogeneous response started during the embryonic development, which makes essential having a dedicated chip that allows including this early developmental stage in the drug assay. Our approach would therefore allow elucidating pharmaceutical or therapeutic responses that so far were still being overlooked.
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In a typical high-throughput drug screening (HTS) process, up to millions of chemical compounds are applied to cells cultured in well plates, aiming to find molecules that exhibit a robust dose-response, as evidenced for example by a fluorescence signal. In high-content screening (HCS), one goes a step further by linking the tested compounds to phenotypic information, obtained, for instance, from microscopic cell images, thereby creating richer data sets that also require more advanced analysis methods. The nematode Caenorhabditis elegans came into the screening picture due to the wide availability of its mutants and human disease models, its relatively easy culture and short life cycle. Being a whole-organism model, it allows drug testing under physiological conditions at multi-tissue levels and provides additional observable phenotypes with respect to cell models, related, for instance, to development, aging, behavior or motility. Worm-based HTS studies in liquid environments on microwell plates have been demonstrated, while microfluidic devices allowed surpassing the performance of plates by enabling more versatile and accurate assays, precise and dynamic dosing of compounds, and readouts down to single-animal resolution. In this review, we discuss microfluidic devices for C. elegans analysis and related studies, published in the period from 2012 to 2017. After an introduction to the different screening approaches, we first focus on microfluidic systems with potential for screening applications. Various enabling technologies, e.g. electrophysiological on-chip recordings or laser axotomy, have been implemented, as well as techniques for reversible worm immobilization and high-resolution imaging, combined with algorithms for automated experimentation and analysis. Several devices for developmental or behavioral assays, and worm sorting based on different phenotypes, have been proposed too. In a subsequent section, we review the application of microfluidic-based systems for medium- and high-throughput screens, including neurobiology and neurodegeneration studies, aging and developmental assays, toxicity and pathogenesis screens, as well as behavioral and motility assays. A thorough analysis of this work reveals a trend towards microfluidic systems more and more capable of offering high-quality analyses of large worm populations, based on multi-phenotypic and/or longitudinal readouts, with clear potential for their application in larger HTS/HCS contexts.
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Caenorhabditis elegans , Ensaios de Triagem em Larga Escala/instrumentação , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Técnicas de Cultura , Desenho de EquipamentoRESUMO
The roundworm Caenorhabditis elegans (C. elegans) is a powerful model organism for addressing fundamental biological questions related to human disease and aging. Its life cycle consists of an embryo stage, four larval stages that can be clearly distinguished by size and different morphological features, and adulthood. Many worm-based bio-assays require stage- or age-synchronized worm populations, for example for studying the life cycle and aging of worms under different pharmacological conditions or to avoid misinterpretation of results due to overlap of stage-specific response in general. Here, we present a new microfluidic approach for size-dependent sorting of C. elegans nematodes on-chip. We take advantage of the external pressure-deformable profile of polydimethylsiloxane (PDMS) transfer channels that connect two on-chip worm chambers. The pressure-controlled effective cross-section of these channels creates adjustable filter structures that can be easily tuned for a specific worm sorting experiment, without changing the design parameters of the device itself. By optimizing the control pressure settings, we can extract larvae of a specific development stage from a mixed worm culture with an efficiency close to 100% and with a throughput of up to 3.5 worms per second. Our approach also allows us to generate mixed populations of larvae of adjacent stages or to adjust their ratio directly in the microfluidic chamber. Moreover, using the same device, we demonstrated extraction of embryos from adult worm populations for subsequent culture of accurately age-synchronized nematode populations or embryo-based assays. Considering that our sorting device is merely based on geometrical parameters and operated by simple fluidic and pressure control, we believe that it has strong potential for use in advanced, automated, microfluidic C. elegans-based assay platforms.
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Caenorhabditis elegans/embriologia , Embrião não Mamífero , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , AnimaisRESUMO
Like other animals, C. elegans nematodes have the ability to socially interact and to communicate through exchange and sensing of small soluble signaling compounds that help them cope with complex environmental conditions. For the time being, worm biocommunication assays are being performed mainly on agar plates; however, microfluidic assays may provide significant advantages compared to traditional methods, such as control of signaling molecule concentrations and gradients or confinement of distinct worm populations in different microcompartments. Here, we propose a microfluidic device for studying signaling via diffusive secreted compounds between two specific C. elegans populations over prolonged durations. In particular, we designed a microfluidic assay to investigate the biological process of male-induced demise, i.e. lifespan shortening and accelerated age-related phenotype alterations, in C. elegans hermaphrodites in the presence of a physically separated male population. For this purpose, male and hermaphrodite worm populations were confined in adjacent microchambers on the chip, whereas molecules secreted by males could be exchanged between both populations by periodically activating the controlled fluidic transfer of µl-volume aliquots of male-conditioned medium. For male-conditioned hermaphrodites, we observed a reduction of 4 days in mean lifespan compared to the non-conditioned on-chip culture. We also observed an enhanced muscle decline, as expressed by a faster decrease in the thrashing frequency and the appearance of vacuolar-like structures indicative of accelerated aging. The chip was placed in an incubator at 20 °C for accurate control of the lifespan assay conditions. An on-demand bacteria feeding protocol was applied, and the worms were observed during long-term on-chip culture over the whole worm lifespan.
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Comunicação Animal , Bioensaio/instrumentação , Caenorhabditis elegans/fisiologia , Animais , Dispositivos Lab-On-A-Chip , Masculino , Fenótipo , ReproduçãoRESUMO
BACKGROUND: While many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions - e.g. neurodegenerative diseases - often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field. RESULTS: We describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms' tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system's ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model. CONCLUSION: Our microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential.
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Caenorhabditis elegans/metabolismo , Superóxido Dismutase/metabolismo , Animais , Modelos Animais de Doenças , Doxiciclina/farmacologia , TemperaturaRESUMO
Phenotyping strategies in simple model organisms such as D. melanogaster and C. elegans are often broadly limited to growth, aging, and fitness. Recently, a number of physical setups and video tracking software suites have been developed to allow for accurate, quantitative, and high-throughput analysis of movement in flies and worms. However, many of these systems require precise experimental setups and/or fixed recording formats. We report here an update to the Parallel Worm Tracker software, which we termed the Movement Tracker. The Movement Tracker allows variable experimental setups to provide cross-platform automated processing of a variety of movement characteristics in both worms and flies and permits the use of simple physical setups that can be readily implemented in any laboratory. This software allows high-throughput processing capabilities and high levels of flexibility in video analysis, providing quantitative movement data on C. elegans and D. melanogaster in a variety of different conditions. © 2016 by John Wiley & Sons, Inc.
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Comportamento Animal/fisiologia , Software , Animais , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/metabolismo , Locomoção/fisiologia , Modelos Animais , Estatística como Assunto/instrumentaçãoRESUMO
Magnetic micro- and nanoparticles ('magnetic beads') have been used to advantage in many microfluidic devices for sensitive antigen (Ag) detection. Today, assays that use as read-out of the signal the number count of immobilized beads on a surface for quantification of a sample's analyte concentration have been among the most sensitive and have allowed protein detection lower than the fgmL(-1) concentration range. Recently, we have proposed in this category a magnetic bead surface coverage assay (Tekin et al., 2013 [1]), in which 'large' (2.8µm) antibody (Ab)-functionalized magnetic beads captured their Ag from a serum and these Ag-carrying beads were subsequently exposed to a surface pattern of fixed 'small' (1.0µm) Ab-coated magnetic beads. When the system was exposed to a magnetic induction field, the magnet dipole attractive interactions between the two bead types were used as a handle to approach both bead surfaces and assist with Ag-Ab immunocomplex formation, while unspecific binding (in absence of an Ag) of a large bead was reduced by exploiting viscous drag flow. The dose-response curve of this type of assay had two remarkable features: (i) its ability to detect an output signal (i.e. bead number count) for very low Ag concentrations, and (ii) an output signal of the assay that was non-linear with respect to Ag concentration. We explain here the observed dose-response curves and show that the type of interactions and the concept of our assay are in favour of detecting the lowest analyte concentrations (where typically either zero or one Ag is carried per large bead), while higher concentrations are less efficiently detected. We propose a random walk process for the Ag-carrying bead over the magnetic landscape of small beads and this model description explains the enhanced overall capture probability of this assay and its particular non-linear dose response curves.