Microfluidic chips provide visual access to in situ soil ecology.
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ABSTRACT:
SUBMITTER: Mafla-Endara PM
PROVIDER: S-EPMC8292388 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.
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Project description:Although microfluidics has shown exciting potential, its broad applications are significantly limited by drawbacks of the materials used to make them. In this work, we present a convenient strategy for fabricating whole-Teflon microfluidic chips with integrated valves that show outstanding inertness to various chemicals and extreme resistance against all solvents. Compared with other microfluidic materials [e.g., poly(dimethylsiloxane) (PDMS)] the whole-Teflon chip has a few more advantages, such as no absorption of small molecules, little adsorption of biomolecules onto channel walls, and no leaching of residue molecules from the material bulk into the solution in the channel. Various biological cells have been cultured in the whole-Teflon channel. Adherent cells can attach to the channel bottom, spread, and proliferate well in the channels (with similar proliferation rate to the cells in PDMS channels with the same dimensions). The moderately good gas permeability of the Teflon materials makes it suitable to culture cells inside the microchannels for a long time.
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Project description:Gene expression and genetic regulatory networks in multi-cellular organisms control complex physiological processes ranging from cellular differentiation to development to aging. Traditional methods to investigate gene expression relationships rely on using bulk, pooled-population assays (e.g. RNA-sequencing and RT-PCR) to compare gene expression levels in hypo- or hyper-morphic mutant animals (e.g. gain-of-function or knockout). This approach is limited, especially in complex gene networks, as these genetic mutations may affect the expressions of related genes in unforseen ways. In contrast, we developed a microfluidic-based pipeline to discover gene relationships in a single genetic background. The microfluidic device provides efficient reagent exchange and the ability to track individual animals. By automating a robust microfluidic reagent exchange strategy, we adapted and validated single molecule fluorescent in situ hybridization (smFISH) on-chip and combined this technology with live-imaging of fluorescent transcriptional reporters. Together, this multi-level information enabled us to quantify a gene expression relationship with single-animal resolution. While this microfluidic-based pipeline is optimized for live-imaging and smFISH C. elegans studies, the strategy is highly-adaptable to other biological models as well as combining other live and end-point biological assays, such as behavior-based toxicology screening and immunohistochemistry.
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Project description:Paper-based microfluidics are an increasingly popular alternative to devices with conventional open channel geometries. The low cost of fabrication and the absence of external instrumentation needed to drive paper microchannels make them especially well suited for medical diagnostics in resource-limited settings. Despite the advantages of paper microfluidics, many assays performed using conventional open channel microfluidics are challenging to translate onto paper, such as bead, emulsion, and cell-based assays. To overcome this challenge, we have developed a hybrid open-channel/paper channel microfluidic device. In this design, wick-driven paper channels control the flow rates within conventional microfluidics. We fabricate these hybrid chips using laser-micromachined polymer sheets and filter paper. In contrast to previous efforts that utilized external, macroscopic paper-based pumps, we integrated micro-scale paper and open channels onto a single chip to control multiple open channels and control complex laminar flow-pattern within individual channels. We demonstrated that flow patterns within the open channels can be quantitatively controlled by modulating the geometry of the paper channels, and that these flow rates agree with Darcy's law. The utility of these hybrid chips, for applications such as bead-, cell-, or emulsion-based assays, was demonstrated by constructing a hybrid chip that hydrodynamically focused micrometer-sized polystyrene beads stably for >10 min, as well as cells, without external instrumentation to drive fluid flow.
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Project description:Monitoring nutrients in the soil can provide valuable information for understanding their spatiotemporal variability and informing precise soil management. Here, we describe an autonomous in situ analyzer for the real-time monitoring of nitrate in soil. The analyzer can sample soil nitrate using either microdialysis or ultrafiltration probes placed within the soil and quantify soil nitrate using droplet microfluidics and colorimetric measurement. Compared with traditional manual sampling and lab analysis, the analyzer features low reagent consumption (96 μL per measurement), low maintenance requirement (monthly), and high measurement frequency (2 or 4 measurements per day), providing nondrifting lab-quality data with errors of less than 10% using a microdialysis probe and 2-3% for ultrafiltration. The analyzer was deployed at both the campus garden and forest for different periods of time, being able to capture changes in free nitrate levels in response to manual perturbation by the addition of nitrate standard solutions and natural perturbation by rainfall events.
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Project description:Flow rates play an important role in microfluidic devices because they affect the transport of chemicals and determine where and when (bio)chemical reactions occur in these devices. Flow rates can conveniently be determined using external peripherals in active microfluidics. However, setting specific flow rates in passive microfluidics is a significant challenge because they are encoded on a design and fabrication level, leaving little freedom to users for adjusting flow rates for specific applications. Here, we present a programmable hydraulic resistor where an array of "electrogates" routes an incoming liquid through a set of resistors to modulate flow rates in microfluidic chips post-fabrication. This approach combines a battery-powered peripheral device with passive capillary-driven microfluidic chips for advanced flow rate control and measurement. We specifically show a programmable hydraulic resistor composed of 7 parallel resistors and 14 electrogates. A peripheral and smartphone application allow a user to activate selected electrogates and resistors, providing 127 (27-1) flow resistance combinations with values spanning on a 500 fold range. The electrogates feature a capillary pinning site (i.e. trench across the flow path) to stop a solution and an electrode, which can be activated in a few ms using a 3?V bias to resume flow based on electrowetting. The hydraulic resistor and microfluidic chip shown here enable flow rates from ~0.09 nL.s-1 up to ~5.66 nL.s-1 with the resistor occupying a footprint of only 15.8?mm2 on a 1 × 2?cm2 microfluidic chip fabricated in silicon. We illustrate how a programmable hydraulic resistor can be used to set flow rate conditions for laminar co-flow of 2 liquids and the enzymatic conversion of a substrate by stationary enzymes (alkaline phosphatase) downstream of the programmable hydraulic resistor.
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Project description:A microfluidic chip with an integrated planar waveguide was fabricated in poly(methyl methacrylate), PMMA, using a single-step, double-sided hot-embossing approach. The waveguide was embedded in air on three sides, the solution being interrogated on the fourth. DNA probes were covalently attached to the waveguide surface by plasma activating the PMMA and the use of carbodiimide coupling chemistry. Successful hybridization events were read using evanescent excitation monitored by an imaging microscope, which offered high spatial resolution (2 microm) and a large field-of-view (20 mm diameter field-of-view), providing imaging of the entire array without scanning. The application of the microfluidic/waveguide assembly was demonstrated by detecting low abundant point mutations; insertion C mutations in BRCA1 genes associated with breast cancer were analyzed using a universal array coupled to an allele-specific ligation assay. DNA probes consisting of amine-terminated oligonucleotides were printed inside the microfluidic channel using a noncontact microspotter. Mutant and wild-type genomic DNAs of BRCA1 were PCR (polymerase chain reaction) amplified, with the amplicons subjected to ligation detection reactions (LDRs). LDR solutions were allowed to flow over the microarray positioned on the polymer waveguide with successful ligation events discerned through fluorescence signatures present at certain locations of the array. The microfluidic/waveguide assembly could detect polymorphisms present at <1% of the total DNA content.
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Project description:In vitro models of human organs must accurately reconstitute oxygen concentrations and gradients that are observed in vivo to mimic gene expression, metabolism, and host-microbiome interactions. Here we describe a simple strategy to achieve physiologically relevant oxygen tension in a two-channel human small intestine-on-a-chip (Intestine Chip) lined with primary human duodenal epithelium and intestinal microvascular endothelium in parallel channels separated by a porous membrane while both channels are perfused with oxygenated medium. This strategy was developed using computer simulations that predicted lowering the oxygen permeability of poly-dimethylsiloxane (PDMS) chips in specified locations using a gas impermeable film will allow the cells to naturally decrease the oxygen concentration through aerobic respiration and reach steady-state oxygen levels <36 mm Hg (<5%) within the epithelial lumen. The approach was experimentally confirmed using chips with embedded oxygen sensors that maintained this stable oxygen gradient. Furthermore, Intestine Chips cultured with this approach supported formation of a villus epithelium interfaced with a continuous endothelium and maintained intestinal barrier integrity for 72 h. This strategy recapitulates in vivo functionality in an efficient, inexpensive, and scalable format that improves the robustness and translatability of Organ Chip technology for studies on microbiome as well as oxygen sensitivity.
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Project description:Liquid filling in microfluidic channels is a complex process that depends on a variety of geometric, operating, and material parameters such as microchannel geometry, flow velocity∕pressure, liquid surface tension, and contact angle of channel surface. Accurate analysis of the filling process can provide key insights into the filling time, air bubble trapping, and dead zone formation, and help evaluate trade-offs among the various design parameters and lead to optimal chip design. However, efficient modeling of liquid filling in complex microfluidic networks continues to be a significant challenge. High-fidelity computational methods, such as the volume of fluid method, are prohibitively expensive from a computational standpoint. Analytical models, on the other hand, are primarily applicable to idealized geometries and, hence, are unable to accurately capture chip level behavior of complex microfluidic systems. This paper presents a parametrized dynamic model for the system-level analysis of liquid filling in three-dimensional (3D) microfluidic networks. In our approach, a complex microfluidic network is deconstructed into a set of commonly used components, such as reservoirs, microchannels, and junctions. The components are then assembled according to their spatial layout and operating rationale to achieve a rapid system-level model. A dynamic model based on the transient momentum equation is developed to track the liquid front in the microchannels. The principle of mass conservation at the junction is used to link the fluidic parameters in the microchannels emanating from the junction. Assembly of these component models yields a set of differential and algebraic equations, which upon integration provides temporal information of the liquid filling process, particularly liquid front propagation (i.e., the arrival time). The models are used to simulate the transient liquid filling process in a variety of microfluidic constructs and in a multiplexer, representing a complex microfluidic network. The accuracy (relative error less than 7%) and orders-of-magnitude speedup (30 000X-4 000 000X) of our system-level models are verified by comparison against 3D high-fidelity numerical studies. Our findings clearly establish the utility of our models and simulation methodology for fast, reliable analysis of liquid filling to guide the design optimization of complex microfluidic networks.
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Project description:Over the past decade, there has been a growth of interest in the translation of microfluidic systems into real-world clinical practice, especially for use in point-of-care or near patient settings. While initial fabrication advances in microfluidics involved mainly the etching of silicon and glass, the economics of scaling of these materials is not amendable for point-of-care usage where single-test applications force cost considerations to be kept low and throughput high. As such, materials base more consistent with point-of-care needs is required. In this manuscript, the fabrication of a hot embossed, through-hole low-density polyethylene ensembles derived from an anisotropically etched silicon wafer is discussed. This semi-opaque polymer that can be easily sterilized and recycled provides low background noise for fluorescence measurements and yields more affordable cost than other thermoplastics commonly used for microfluidic applications such as cyclic olefin copolymer (COC). To fabrication through-hole microchips from this alternative material for microfluidics, a fabrication technique that uses a high-temperature, high-pressure resistant mold is described. This aluminum-based epoxy mold, serving as the positive master mold for embossing, is casted over etched arrays of pyramidal pits in a silicon wafer. Methods of surface treatment of the wafer prior to casting and PDMS casting of the epoxy are discussed to preserve the silicon wafer for future use. Changes in the thickness of polyethylene are observed for varying embossing temperatures. The methodology described herein can quickly fabricate 20 disposable, single use chips in less than 30 min with the ability to scale up 4 times by using multiple molds simultaneously. When coupled as a platform supporting porous bead sensors, as in the recently developed Programmable Bio-Nano-Chip, this bead chip system can achieve limits of detection, for the cardiac biomarker C-reactive protein, of 0.3 ng/mL, thereby demonstrating that the approach is compatible with high performance, real-world clinical measurements in the context of point-of-care testing.
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