Microfluidic systems for infectious disease diagnostics.

Microorganisms, encompassing both uni- and multicellular entities, exhibit remarkable diversity as omnipresent life forms in nature. They play a pivotal role by supplying essential components for sustaining biological processes across diverse ecosystems, including higher host organisms. The complex interactions within the human gut microbiota are crucial for metabolic functions, immune responses, and biochemical signalling, particularly through the gut-brain axis. Viruses also play important roles in biological processes, for example by increasing genetic diversity through horizontal gene transfer when replicating inside living cells. On the other hand, infection of the human body by microbiological agents may lead to severe physiological disorders and diseases. Infectious diseases pose a significant burden on global healthcare systems, characterized by substantial variations in the epidemiological landscape. Fast spreading antibiotic resistance or uncontrolled outbreaks of communicable diseases are major challenges at present. Furthermore, delivering field-proven point-of-care diagnostic tools to the most severely affected populations in low-resource settings is particularly important and challenging. New paradigms and technological approaches enabling rapid and informed disease management need to be implemented. In this respect, infectious disease diagnostics taking advantage of microfluidic systems combined with integrated biosensor-based pathogen detection offers a host of innovative and promising solutions. In this review, we aim to outline recent activities and progress in the development of microfluidic diagnostic tools. Our literature research mainly covers the last 5 years. We will follow a classification scheme based on the human body systems primarily involved at the clinical level or on specific pathogen transmission modes. Important diseases, such as tuberculosis and malaria, will be addressed more extensively.

Difference in Intestine Content of Caenorhabditis elegans When Fed on Non-Pathogenic or Pathogenic Bacteria.

We investigated the bacterial food digestion and accumulation in wild-type adult Caenorhabditis elegans (C. elegans) worms that have fed on either non-pathogenic RFP-expressing Escherichia coli (E. coli) OP50 or pathogenic-RFP-expressing Pseudomonas aeruginosa (P. aeruginosa) PAO1 during the first 4 days of adulthood. Once the worms had completed their planned feeding cycles, they were loaded on microfluidic chips, where they were fixed to allow high-resolution z-stack fluorescence imaging of their intestines utilizing a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution oil-immersion objective (60×). IMARIS software was used to visualize and analyze the obtained images, resulting in the production of three-dimensional constructs of the intestinal bacterial load. We discovered two distinct patterns for the bacteria-derived fluorescence signal in the intestine: (i) individual fluorescent spots, originating from intact bacteria, were present in the fluorescent E. coli-OP50-fed worms, and (ii) individual fluorescent spots (originating from intact bacteria) were dispersed in large regions of diffuse fluorescence (RDF), originating from disrupted bacteria, in fluorescent P. aeruginosa-PAO1-fed worms. We performed a semi-automated single-worm-resolution quantitative analysis of the intestinal bacterial load, which showed that the intestinal bacterial load generally increases with age of the worms, but more rapidly for the fluorescent P. aeruginosa-PAO1-fed worms.

Acoustofluidic large-scale mixing for enhanced microfluidic immunostaining for tissue diagnostics.

The usage of microfluidics for automated and fast immunoassays has gained a lot of interest in the last decades. This integration comes with certain challenges, like the reconciliation of laminar flow patterns of micro-scale systems with diffusion-limited mass transport. Several methods have been investigated to enhance microfluidic mixing in microsystems, including acoustic-based fluidic streaming. Here, we report both by numerical simulation and experiments on the beneficiary effect of acoustic agitation on the uniformity of immunostaining in large-size and thin microfluidic chambers. Moreover, we investigate by numerical simulation the impact of reducing the incubation times and the concentrations of the biochemical detection reagents on the obtained immunoassay signal. Finally, acoustofluidic mixing was successfully used to reduce by 80% the incubation time of the Her2 (human epidermal growth factor receptor 2) and CK (cytokeratins) biomarkers for the spatial immunostaining of breast cancer cell pellets, or reducing their concentration by 66% and achieving a higher signal-to-background ratio than comparable spatially resolved immunostaining with static incubation.

High-resolution imaging and analysis of the intestinal bacterial load of Caenorhabditis elegans during early adulthood.

We study the presence within the worm Caenorhabditis elegans (C. elegans) of a fluorescent strain of the worm's bacterial food (Escherichia coli (E. coli) OP50) during early adulthood. Use of a microfluidic chip based on a thin glass coverslip substrate allows investigation of the intestinal bacterial load using a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution objective (60×). High-resolution z-stack fluorescence images of the gut bacteria in adult worms, which were loaded in the microfluidic chip and subsequently fixed, were analyzed using IMARIS software and 3D reconstructions of the intestinal bacterial load in the worms were obtained. We present an automated bivariate histogram analysis of the volumes and intensities of the bacterial spots for each worm and find that, as the worms age, the bacterial load in their hindguts increases. We show the advantage of single-worm resolution automated analysis for bacterial load studies and anticipate that the methods described in our work can be easily implemented in existing microfluidic solutions to enable thorough studies of bacterial proliferation.

Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle.

Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.

Correction: Bubble-enhanced ultrasonic microfluidic chip for rapid DNA fragmentation.

Correction for 'Bubble-enhanced ultrasonic microfluidic chip for rapid DNA fragmentation' by Lin Sun et al., Lab Chip, 2022, 22, 560-572, https://doi.org/10.1039/D1LC00933H.

Efficient AC electrothermal flow (ACET) on-chip for enhanced immunoassays.

Biochemical reaction rates in microfluidic systems are known to be limited by the diffusional transport of reagents, leading often to lowered sensitivity and/or longer detection times in immunoassays. Several methods, including electrically powering electrodes to generate AC electrothermal flow (ACET) on-chip, have been adopted to enhance the mass transport of the reagents and improve microfluidic mixing. Here, we report a novel ACET electrode design concept for generating in-plane microfluidic mixing vortices that act over a large volume close to the reaction surface of interest. This is different from the traditional ACET parallel electrode design that provides rather local vertical mixing vortices directly above the electrodes. Both numerical simulation and experimental studies were performed to validate the new design. Moreover, numerical simulation was carried out to show the effects of experimental factors such as the reaction kinetics (association constant) and the reagent concentration on the ACET-enhanced surface-based assays. As a proof of concept, the new design for the ACET-enhanced immunoassays was used to improve the immunostaining signal of the HER2 (human epidermal growth factor receptor 2) cancer biomarker on breast cancer cells. Finally, the concept of scaling up the design has been validated by experiments (immunoassays on breast cancer cells for different ACET power and different assay times). In particular, we show that larger ACET in-plane designs can agitate and mix the fluid over large microfluidic volumes, which further enhances the immunoassay's output. We have achieved a 6-times enhancement in the assay signal with a 75% reduction in assay time.

Polydimethylsiloxane microstructure-induced acoustic streaming for enhanced ultrasonic DNA fragmentation on a microfluidic chip.

Next-generation sequencing (NGS) is an essential technology for DNA identification in genomic research. DNA fragmentation is a critical step for NGS and doing this on-chip is of great interest for future integrated genomic solutions. Here we demonstrate fast acoustofluidic DNA fragmentation via ultrasound-actuated elastic polydimethylsiloxane (PDMS) microstructures that induce acoustic streaming and associated shear forces when placed in the field of an ultrasonic transducer. Indeed, acoustic streaming locally generates high tensile stresses that can mechanically stretch and break DNA molecule chains. The improvement in efficiency of the on-chip DNA fragmentation is due to the synergistic effect of these tensile stresses and ultrasonic cavitation phenomena. We tested these microstructure-induced effects in a DNA-containing microfluidic channel both experimentally and by simulation. The DNA fragmentation process was evaluated by measuring the change in the DNA fragment size over time. The chip works well with both long and short DNA chains; in particular, purified lambda (λ) DNA was cut from 48.5 kbp to 3 kbp in one minute with selected microstructures and further down to 300 bp within two and a half minutes. The fragment size of mouse genomic DNA was reduced from 1.4 kbp to 400 bp in one minute and then to 200 bp in two and a half minutes. The DNA fragmentation efficiency of the chip equipped with the PDMS microstructures was twice that of the chip without the microstructures. Exhaustive comparison shows that the on-chip fragmentation performance reaches the level of high-end professional standards. Recently, DNA fragmentation was shown to be enhanced using vibrating air microbubbles when the chip was placed in an acoustic field. We think the microbubble-free microstructure-based device we present is easier to operate and more reliable, as it avoids microbubble preparation and maintenance, while showing high DNA fragmentation performance.

The enhancement of DNA fragmentation in a bench top ultrasonic water bath with needle-induced air bubbles: Simulation and experimental investigation.

Shearing DNA to a certain size is the first step in many medical and biological applications, especially in next-generation gene sequencing technology. In this article, we introduced a highly efficient ultrasonic DNA fragmentation method enhanced by needle-induced air bubbles, which is easy to operate with high throughput. The principle of the bubble-enhanced sonication system is introduced and verified by flow field and acoustic simulations and experiments. Lambda DNA long chains and mouse genomic DNA short chains are used in the experiments for testing the performance of the bubble-enhanced ultrasonic DNA fragmentation system. Air bubbles are an effective enhancement agent for ultrasonic DNA fragmentation; they can obviously improve the sound pressure level in the whole solution, thus, achieving better absorption of ultrasound energy. Growing bubbles also have a stretched function on DNA molecule chains and form a huge pressure gradient in the solution, which is beneficial to DNA fragmentation. Purified λDNA is cut from 48.5 to 2 kbp in 5 min and cut to 300 bp in 30 min. Mouse genomic DNA (≈1400 bp) decreases to 400 bp in 5 min and then reduces to 200 bp in 30 min. This bubble-enhanced ultrasonic method enables widespread access to genomic DNA fragmentation in a standard ultrasonic water bath for many virus sequencing demands even without good medical facilities.

Bubble-enhanced ultrasonic microfluidic chip for rapid DNA fragmentation.

DNA fragmentation is an essential process in developing genetic sequencing strategies, genetic research, as well as for the diagnosis of diseases with a genetic signature like cancer. Efficient on-chip DNA fragmentation protocols would be beneficial to process integration and open new opportunities for microfluidics in genetic applications. Here we present an acoustic microfluidic chip comprising an array of ultrasound-actuated microbubbles located at dedicated positions adjacent to a channel containing the DNA sample solution. The efficiency of the on-chip DNA fragmentation process arises mainly from tensile forces generated by acoustic streaming near the oscillating bubble interfaces, as well as a synergistic effect of streaming stress and ultrasonic cavitation. Acoustic microstreaming and the pressure distribution in the DNA channel were assessed by finite element simulation. We characterized the bubble-enhanced effect by measuring gene fragment size distributions with respect to different ultrasound parameters. For optimized on-chip conditions, purified lambda (λ) DNA (48.5 kbp) could be disrupted to fragments with an average size of 2 kbp after 30 s and down to 300 bp after 90 s. Mouse genomic DNA (1.4 kbp) fragmentation size decreased to 500 bp in 30 s and reduced further to 250 bp in 90 s. Bubble-induced fragmentation was more than 3 times faster than without bubbles. On-chip performance and process yield were found to be comparable to a sophisticated high-end commercial system. In this view, our new bubble-enhanced microfluidic approach is a promising tool for current and next generation sequencing platforms with high efficiency and good capacity. Moreover, the availability of an efficient on-chip DNA fragmentation process opens perspectives for implementing full molecular protocols on a single microfluidic platform.

Ripening of two-dimensional colloidal CdSe nanocrystals into zero-dimensional nanodots.

Understanding the ripening of two-dimensional (2D) colloidal nanocrystals (NCs) is important for the controllable synthesis of NCs with desired morphology and properties. In this study, we systematically investigate the ripening behavior of the 2D CdSe NCs in the presence of a short-chain acetate ligand and a long-chain oleate ligand. We find that a low acetate/oleate ratio, a low Cd/Se ratio, and a low monomer concentration help in the ripening of the 2D NCs to form 0D NCs. Moreover, a porous nanosheet intermediate is observed when there is a high Cd/Se ratio, whereas in the case of a low Cd/Se ratio, the ripening starts from the edge of the nanosheets, resulting in a saw-like nanosheet intermediate. These findings provide necessary insights into the growth and ripening of 2D CdSe NCs that allow for the controlled synthesis of 0D and 2D CdSe NCs.

Effect of inoculum size and antibiotics on bacterial traveling bands in a thin microchannel defined by optical adhesive.

Phenotypic diversity in bacterial flagella-induced motility leads to complex collective swimming patterns, appearing as traveling bands with transient locally enhanced cell densities. Traveling bands are known to be a bacterial chemotactic response to self-generated nutrient gradients during growth in resource-limited microenvironments. In this work, we studied different parameters of Escherichia coli (E. coli) collective migration, in particular the quantity of bacteria introduced initially in a microfluidic chip (inoculum size) and their exposure to antibiotics (ampicillin, ciprofloxacin, and gentamicin). We developed a hybrid polymer-glass chip with an intermediate optical adhesive layer featuring the microfluidic channel, enabling high-content imaging of the migration dynamics in a single bacterial layer, i.e., bacteria are confined in a quasi-2D space that is fully observable with a high-magnification microscope objective. On-chip bacterial motility and traveling band analysis was performed based on individual bacterial trajectories by means of custom-developed algorithms. Quantifications of swimming speed, tumble bias and effective diffusion properties allowed the assessment of phenotypic heterogeneity, resulting in variations in transient cell density distributions and swimming performance. We found that incubation of isogeneic E. coli with different inoculum sizes eventually generated different swimming phenotype distributions. Interestingly, incubation with antimicrobials promoted bacterial chemotaxis in specific cases, despite growth inhibition. Moreover, E. coli filamentation in the presence of antibiotics was assessed, and the impact on motility was evaluated. We propose that the observation of traveling bands can be explored as an alternative for fast antimicrobial susceptibility testing.

An In Vivo Microfluidic Study of Bacterial Load Dynamics and Absorption in the C. elegans Intestine.

Caenorhabditiselegans (C. elegans) has gained importance as a model for studying host-microbiota interactions and bacterial infections related to human pathogens. Assessing the fate of ingested bacteria in the worm's intestine is therefore of great interest, in particular with respect to normal bacterial digestion or intestinal colonization by pathogens. Here, we report an in vivo study of bacteria in the gut of C. elegans. We take advantage of a polydimethylsiloxane (PDMS) microfluidic device enabling passive immobilization of adult worms under physiological conditions. Non-pathogenic Escherichia coli (E. coli) bacteria expressing either pH-sensitive or pH-insensitive fluorescence reporters as well as fluorescently marked indigestible microbeads were used for the different assays. Dynamic fluorescence patterns of the bacterial load in the worm gut were conveniently monitored by time-lapse imaging. Cyclic motion of the bacterial load due to peristaltic activity of the gut was observed and biochemical digestion of E. coli was characterized by high-resolution fluorescence imaging of the worm's intestine. We could discriminate between individual intact bacteria and diffuse signals related to disrupted bacteria that can be digested. From the decay of the diffuse fluorescent signal, we determined a digestion time constant of 14 ± 4 s. In order to evaluate the possibility to perform infection assays with our platform, immobilized C. elegans worms were fed pathogenic Mycobacterium marinum (M. marinum) bacteria. We analyzed bacterial fate and accumulation in the gut of N2 worms and mitochondrial stress response in a hsp-6::gfp mutant.

Antimicrobial susceptibility testing by measuring bacterial oxygen consumption on an integrated platform.

Cellular respiration is a fundamental feature of metabolic activity and oxygen consumption can be considered as a reliable indicator of bacterial aerobic respiration, including for facultative anaerobic bacteria like E. coli. Addressing the emerging global health challenge of antimicrobial resistance, we performed antimicrobial susceptibility testing using the bacterial oxygen consumption rate (OCR) as a phenotypic indicator. We demonstrated that microbial exposure to antibiotics showed systematic OCR variations, which enabled determining minimum inhibitory concentrations for three clinically relevant antibiotics, ampicillin, ciprofloxacin, and gentamicin, within a few hours. Our study was performed by using photoluminescence-based oxygen sensing in a microchamber format, which enabled reducing the sample volume to a few hundred microliters. OCR modeling based on exponential bacterial growth allowed estimating the bacterial doubling time for various culture conditions (different types of media, different culture temperature and antibiotic concentrations). Furthermore, correlating metabolic heat production data, as obtained by nanocalorimetry in the same type of microchamber, and OCR measurements provided further insight on the actual metabolic state and activity of a microbial sample. This approach represents a new path towards more comprehensive microbiological studies performed on integrated miniaturized systems.

Fast antimicrobial susceptibility testing on Escherichia coli by metabolic heat nanocalorimetry.

Fast spreading of antimicrobial resistance is now considered a major global health threat. New technologies are required, enabling rapid diagnostics of bacterial infection combined with fast antimicrobial susceptibility testing (AST) for evaluating the efficiency and dosage of antimicrobial compounds in vitro. This work presents an integrated chip-based isothermal nanocalorimetry platform for direct microbial metabolic heat measurements and evaluates its potential for fast AST. Direct detection of the bacteria-generated heat allows monitoring of metabolic activity and antimicrobial action at subinhibitory concentrations in real-time. The high heat sensitivity of the platform enables bacterial growth detection within only a few hours of incubation, whereas growth inhibition upon administration of antibiotics is revealed by a decrease or the absence of the heat signal. Antimicrobial stress results in lag phase extension and metabolic energy spilling. Oxygen consumption and optical density measurements provide a more holistic insight of the metabolic state and the evolution of bacterial biomass. As a proof-of-concept, a metabolic heat-based AST study on Escherichia coli as model organism with 3 clinically relevant antibiotics is performed and the minimum inhibitory concentrations are determined.

An in vivo microfluidic study of bacterial transit in C. elegans nematodes.

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.

Force microscopy of the Caenorhabditis elegans embryonic eggshell.

Assays focusing on emerging biological phenomena in an animal's life can be performed during embryogenesis. While the embryo of Caenorhabditis elegans has been extensively studied, its biomechanical properties are largely unknown. Here, we demonstrate that cellular force microscopy (CFM), a recently developed technique that combines micro-indentation with high resolution force sensing approaching that of atomic force microscopy, can be successfully applied to C. elegans embryos. We performed, for the first time, a quantitative study of the mechanical properties of the eggshell of living C. elegans embryos and demonstrate the capability of the system to detect alterations of its mechanical parameters and shell defects upon chemical treatments. In addition to investigating natural eggshells, we applied different eggshell treatments, i.e., exposure to sodium hypochlorite and chitinase solutions, respectively, that selectively modified the multilayer eggshell structure, in order to evaluate the impact of the different layers on the mechanical integrity of the embryo. Finite element method simulations based on a simple embryo model were used to extract characteristic eggshell parameters from the experimental micro-indentation force-displacement curves. We found a strong correlation between the severity of the chemical treatment and the rigidity of the shell. Furthermore, our results showed, in contrast to previous assumptions, that short bleach treatments not only selectively remove the outermost vitelline layer of the eggshell, but also significantly degenerate the underlying chitin layer, which is primarily responsible for the mechanical stability of the egg.

Automated phenotyping of Caenorhabditis elegans embryos with a high-throughput-screening microfluidic platform.

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.

PDMS filter structures for size-dependent larval sorting and on-chip egg extraction of C. elegans.

C. elegans-based assays require age-synchronized populations prior to experimentation to achieve standardized sets of worm populations, due to which age-induced heterogeneous phenotyping effects can be avoided. There have been several approaches to synchronize populations of C. elegans at certain larval stages; however, many of these methods are tedious, complex and have low throughput. In this work, we demonstrate a polydimethylsiloxane (PDMS) microfluidic filtering device for high-throughput, efficient, and extremely rapid sorting of mixed larval populations of C. elegans. Our device consists of three plasma-activated and bonded PDMS parts and permits sorting of mixed populations of two consecutive larval stages in a matter of minutes. After sorting, we also retain the remaining larval stage of the initially mixed worm population on the chip, thereby enabling collection of the two sorted larval populations from the device. We demonstrated that the target larvae could be collected from a mixed worm population by cascading these devices. Our approach is based on only passive hydrodynamics filter structures, resulting in a user-friendly and reusable tool. In addition, we employed the equivalent of a standard bleaching procedure that is practiced in standard worm culture on agar plates for embryo harvesting on our chip, and we demonstrated rapid egg extraction and subsequent harvesting of a synchronized L1 larvae population.

Microfluidic system for Caenorhabditis elegans culture and oxygen consumption rate measurements.

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.