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At least 127 records · Page 7

Microfluidics Synthesis of Gene Silencing Cubosomes

The success of gene technologies hinges on our ability to engineer superior encapsulation and delivery vectors. Cubosomes are lipid-based nanoparticles where membranes, instead of enveloping into classic liposomes, intertwine into complex arrays of pores well-ordered in a cubic lattice. These complex nanoparticles encapsulate large contents of siRNA compared to a liposomal analogue. Importantly, the membranes that form cubosomes have intrinsic fusogenic properties that promote fast endosomal escape. Despite the great potential, traditional routes of forming cubosomes lead to particle sizes too large to fulfill the state-of-the art requirements of delivery vectors. To overcome this challenge, in this work we utilize a microfluidic nanomanufacturing device to synthesize cubosomes and siRNA-loaded cubosomes, termed cuboplexes. Utilizing cryogenic TEM and small angle X-ray scattering we elucidate the time-resolved mechanisms in which microfluidic devices allow the production of small cubosomes and cuboplexes (75 nm) that outperform commercially available delivery vectors, as well as liposome-based systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices

The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. Lastly, these methods should be applicable to a wide range of membrane proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Design, fabrication and test of a pneumatically controlled, renewable, microfluidic bead trapping device for sequential injection analysis applications

This paper describes the design, fabrication, and testing of a pneumatically controlled,renewable, microfluidic device for conducting bead-based assays in an automated sequential injection analysis system. The device used a “brick wall”-like pillar array (pillar size: 20 μm length X 50 μm width X 45 μm height) with 5 μm gaps between the pillars serving as the micro filter. The flow channel where bead trapping occurred is 500 μm wide X 75 μm deep. An elastomeric membrane and an air chamber were located underneath the flow channel. By applying pressure to the air chamber, the membrane is deformed and pushed upward against the filter structure. This effectively traps beads larger than 5 μm and creates a “bed” or micro column of beads that can be perfused and washed with liquid samples and reagents. Upon completion of the assay process, the pressure is released and the beads are flushed out from underneath the filter structure to renew the device. Mouse IgG was used as a model analyte to test the feasibility of using the proposed device for immunoassay applications. Resulting microbeads from an on-chip fluorescent immunoassay were individually examined using flow cytometry. The results show that the fluorescence signal intensity distribution is fairly narrow indicating high chemical reaction uniformity among the beads population. Electrochemical onchip assay was also conducted. A detection limit of 0.1 ng/mL1 ppb was achieved and good device reliability and repeatability were demonstrated. The novel microfluidic-based beadstrapping device thus opens up a new pathway to design micro-bead based biosensor immunoassays for clinical and othervarious applications.

beads trapping↗

Ionic electroactive polymer actuators as active microfluidic mixers

On-chip sample processing is integral to the continued development of lab-on-a-chip devices for various applications. An active microfluidic mixer prototype is proposed using ionic electroactive polymer actuators (IEAPAs) as artificial cilia. A proof-of-concept experiment was performed in which the actuators were shown to produce localized flow pattern disruptions in the laminar flow regime. Suggestions for further engineering and optimization of a scaled-down, complete device are provided. Furthermore, the device in its current state of development necessitates further engineering, the use of IEAPAs addresses issues currently associated with the use of electromechanical actuators as active microfluidic mixers and may prove to be a useful alternative to other similar materials.

36 MATERIALS SCIENCE↗

Development and testing of a novel micro-Raman probe and application of calibration transfer method for the quantitative analysis of microfluidic nitric acid streams

To simplify and improve the safety of reprocessing used nuclear fuel, an initial assessment was made of Raman microscopy applied to microfluidic volumes with a view toward the on-line spectroscopic measurement of highly radioactive solutions. This study compares a microscopic Raman probe (excitation focal point diameter 70 µm) to a larger, well studied probe (excitation focal point diameter 125 µm) used in prior investigations. This was done by chemometrically modeling and predicting concentrations of HNO3 solutions (0 M to 8 M) as they flowed through microfluidic cells based upon spectra from each probe. Spectra recorded for each probe using the same static HNO3 solution set contained in 2 dram glass vials were used as training sets to produce models for the respective probes. Modeling required baseline, normalization and smoothing preprocessing to compensate for a reduced path length between the static glass vial training set (4 cm) and the reduced path length flow cell (1 cm), wide ranging solution concentrations, and the associated non-linear spectral changes, and abrupt and uneven concentration changes of flowing solutions. The micro-Raman probe is able to produce spectra that may be analyzed chemometrically to accurately predict the concentration of flowing HNO3 solutions down to microliter volumes. Based upon RMSECV and RMSEP modeling statistics concentration predictions of the micro-Raman probe are comparable to those obtained for a macro-Raman on identical samples.

microscopic Raman probe, MicroRaman, nitric acid, ↗

All polymer microfluidic chips—A fixed target sample delivery workhorse for serial crystallography

The development of x-ray free electron laser (XFEL) light sources and serial crystallography methodologies has led to a revolution in protein crystallography, enabling the determination of previously unobtainable protein structures and near-atomic resolution of otherwise poorly diffracting protein crystals. However, to utilize XFEL sources efficiently demands the continuous, rapid delivery of a large number of difficult-to-handle microcrystals to the x-ray beam. A recently developed fixed-target system, in which crystals of interest are enclosed within a sample holder, which is rastered through the x-ray beam, is discussed in detail in this Perspective. The fixed target is easy to use, maintains sample hydration, and can be readily modified to allow a broad range of sample types and different beamline requirements. Recent innovations demonstrate the potential of such microfluidic-based fixed targets to be an all-around “workhorse” for serial crystallography measurements. In conclusion, this Perspective will summarize recent advancements in microfluidic fixed targets for serial crystallography, examine needs for future development, and guide users in designing, choosing, and utilizing a fixed-target sample delivery device for their system.

59 BASIC BIOLOGICAL SCIENCES↗

Wetting transition and fluid trapping in a microfluidic fracture

Immiscible fluid–fluid displacement in confined geometries is a fundamental process occurring in many natural phenomena and technological applications, from geological CO 2 sequestration to microfluidics. Due to the interactions between the fluids and the solid walls, fluid invasion undergoes a wetting transition from complete displacement at low displacement rates to leaving a film of the defending fluid on the confining surfaces at high displacement rates. While most real surfaces are rough, fundamental questions remain about the type of fluid–fluid displacement that can emerge in a confined, rough geometry. Here, we study immiscible displacement in a microfluidic device with a precisely controlled structured surface as an analogue for a rough fracture. We analyze the influence of the degree of surface roughness on the wetting transition and the formation of thin films of the defending liquid. We show experimentally, and rationalize theoretically, that roughness affects both the stability and dewetting dynamics of thin films, leading to distinct late-time morphologies of the undisplaced (trapped) fluid. Finally, we discuss the implications of our observations for geologic and technological applications.

42 ENGINEERING↗

Multimodal Microchannel and Nanowell-Based Microfluidic Platforms for Bioimaging

Modern live-cell imaging approaches permit real-time visualization of biological processes. However, limitations for unicellular organism trapping, culturing and long-term imaging can preclude complete understanding of how such microorganisms respond to perturbations in their local environment or linking single-cell variability to whole population dynamics. We have developed microfluidic platforms to overcome prior technical bottlenecks to allow both chemostat and compartmentalized cellular growth conditions using the same device. Additionally, a nanowell-based platform enables a high throughput approach to scale up compartmentalized imaging optimized within the microfluidic device. These channel and nanowell platforms are complementary, and both provide fine control over the local environment as well as the ability to add/replace media components at any experimental time point.

bioimaging↗

Nanofluidic interfaces in microfluidic networks

The integration of nano- and microfluidic technologies enables the construction of tunable interfaces to physical and biological systems across relevant length scales. The ability to perform chemical manipulations of miniscule sample volumes is greatly enhanced through these technologies and extends the ability to manipulate and sample the local fluidic environments at subcellular, cellular and community or tissue scales. Here we describe the development of a flexible surface micromachining process for the creation of nanofluidic channel arrays integrated within SU-8 microfluidic networks. The use of a semi-porous, silicon rich, silicon nitride structural layer allows rapid release of the sacrificial silicon dioxide during the nanochannel fabrication. Nanochannel openings that form the interface to biological samples are customized using focused ion beam milling. The compatibility of these interfaces with on-chip microbial culture is demonstrated.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Dataset for "A Microfluidic Spore Chamber for Long-Term Imaging of Single-Spore Hyphal Development"

Understanding the life cycle of fungal spores is essential for elucidating their roles in pathogenesis, dispersal, and survival. However, studying spore development under controlled, spatially defined conditions remains challenging. Here, we present the Spore Chamber, a custom-built microfluidic platform engineered for parallel trapping and long-term imaging of individual spores under defined media conditions, enabling real-time visualization of hyphal development. Using Aspergillus fumigatus as a model organism, we demonstrate that sparse trapping of individual spores within size-matched trap geometries enables long-term time-lapse imaging of key developmental stages, including germination, polarized hyphal elongation, branching, and conidiophore formation. To assess the device’s capacity to resolve morphogenetic responses to exogenous signals, we introduced lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (COs). Rhizobium-derived, non-sulfated LCO (nsLCO) mixtures induced enhanced secondary branching (hyperbranching), a response not previously reported in A. fumigatus under these signal conditions, to our knowledge, whereas sulfated LCOs and CO4 did not significantly alter branching patterns. In addition, long-term confinement and imaging revealed rare developmental morphologies previously described primarily in mutant strains, including split conidiophore formation, elongated phialides, microcyclic conidiation, and chlamydospore development. Together, these results establish the Spore Chamber as a targeted microfluidic platform for single-spore phenotyping and long-term developmental analysis, with applications in fungal biology, chemical signaling studies, and host–microbe interaction research. Videos of the observed phenomena are included in this data set.

59 BASIC BIOLOGICAL SCIENCES↗

Microfluidics and Metabolomics Reveal Symbiotic Bacterial–Fungal Interactions Between Mortierella elongata and Burkholderia Include Metabolite Exchange

We identified two poplar ( Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other’s growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

59 BASIC BIOLOGICAL SCIENCES↗

An Incompressible, Depth-Averaged Lattice Boltzmann Method for Liquid Flow in Microfluidic Devices with Variable Aperture

Two-dimensional (2D) pore-scale models have successfully simulated microfluidic experiments of aqueous-phase flow with mixing-controlled reactions in devices with small aperture. A standard 2D model is not generally appropriate when the presence of mineral precipitate or biomass creates complex and irregular three-dimensional (3D) pore geometries. We modify the 2D lattice Boltzmann method (LBM) to incorporate viscous drag from the top and bottom microfluidic device (micromodel) surfaces, typically excluded in a 2D model. Viscous drag from these surfaces can be approximated by uniformly scaling a steady-state 2D velocity field at low Reynolds number. We demonstrate increased accuracy by approximating the viscous drag with an analytically-derived body force which assumes a local parabolic velocity profile across the micromodel depth. Accuracy of the generated 2D velocity field and simulation permeability have not been evaluated in geometries with variable aperture. We obtain permeabilities within approximately 10% error and accurate streamlines from the proposed 2D method relative to results obtained from 3D simulations. Additionally, the proposed method requires a CPU run time approximately 40 times less than a standard 3D method, representing a significant computational benefit for permeability calculations.

58 GEOSCIENCES↗

Examination of Haines Jump in Microfluidic Experiments via Evolution Graphs and Interface Tracking

This work examines a type of rapid pore-filling event in multiphase flow through permeable media that is better known as Haines Jump. While existing microfluidic experiments on Haines Jump mostly seek to maintain quasi-steady states through very low bulk flow rates over long periods of time, this work explores the combined use of a highly structured microscale transport network, high-speed fluorescent microscopy, displacement front segmentation algorithms, and a tracking algorithm to build evolution graphs that track displacement fronts as they evolve through high-speed video recording. The resulting evolution graph allows the segmentation of a high-speed recording in both space and time, potentially facilitating topology-cognitive computation on the transport network. Occurrences of Haines Jump are identified in the microfluidic displacement experiments and their significance in bulk flow rates is qualitatively analyzed. The bulk flow rate has little effect on the significance of Haines Jump during merging and splitting, but large bulk flow rates may obscure small bursts at the narrowest part of the throat.

permeable media↗

Stamping Nanoparticles onto the Electrode for Rapid Electrochemical Analysis in Microfluidics

Electrochemical analysis is an efficient way to study various materials. However, nanoparticles are challenging due to the difficulty in fabricating a uniform electrode containing nanoparticles. We developed novel approaches to incorporate nanoparticles as a working electrode (WE) in a three-electrode microfluidic electrochemical cell. Specifically, conductive epoxy was used as a medium for direct application of nanoparticles onto the electrode surface. Three approaches in this work were illustrated, including sequence stamping, mix stamping, and droplet stamping. Shadow masking was used to form the conductive structure in the WE surface on a thin silicon nitride (SiN) membrane. Two types of nanomaterials, namely cerium oxide (CeO2) and graphite, were chosen as representative nanoparticles. The as-fabricated electrodes with attached particles were characterized using atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Electrochemical analysis was performed to verify the feasibility of these nanoparticles as electrodes. Nanomaterials can be quickly assessed for their electrochemical properties using these new electrode fabrication methods in a microfluidic cell, offering a passport for rapid nanomaterial electrochemical analysis in the future.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Identifying Candidate Biomarkers of Ionizing Radiation in Human Pulmonary Microvascular Lumens Using Microfluidics—A Pilot Study

The microvasculature system is critical for the delivery and removal of key nutrients and waste products and is significantly damaged by ionizing radiation. Single-cell capillaries and microvasculature structures are the primary cause of circulatory dysfunction, one that results in morbidities leading to progressive tissue and organ failure and premature death. Identifying tissue-specific biomarkers that are predictive of the extent of tissue and organ damage will aid in developing medical countermeasures for treating individuals exposed to ionizing radiation. In this pilot study, we developed and tested a 17 µL human-derived microvascular microfluidic lumen for identifying candidate biomarkers of ionizing radiation exposure. Through mass-spectrometry-based proteomics, we detected 35 proteins that may be candidate early biomarkers of ionizing radiation exposure. This pilot study demonstrates the feasibility of using humanized microfluidic and organ-on-a-chip systems for biomarker discovery studies. A more elaborate study of sufficient statistical power is needed to identify candidate biomarkers and test medical countermeasures of ionizing radiation.

59 BASIC BIOLOGICAL SCIENCES↗

Rapid Protein Separations in Microfluidic Devices

This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

MICROFLUIDICS↗

Advancing Small Satellite Electronics Heritage for Microfluidic Biological Experiments

DLR's Eu:CROPIS (Euglena and Combined Regenerative Organic-Food Production in Space) mission, launching in 2017, will carry multiple biological payloads into a sun-synchronous orbit, including NASA Ames' PowerCell experiment. PowerCell will attempt to characterize the viability of synthetic biology at micro-g, Lunar, and Martian gravity levels. PowerCell experiment requirements demand an electronic system similar to previous microfluidic biology payloads, but with an expanded feature set. As such, the system was based on PharmaSat (Diaz-Aguado et al. 2009), a previous successful biology payload from NASA Ames, and improved upon. Newer, more miniaturized electronics allow for greater capability with a lower part count and smaller size. Two identical PowerCell enclosures will fly. Each enclosure contains two separate and identical experiments with a 48-segment optical density measurement system, grow light system, microfluidic system for nutrient delivery and waste flushing, plus thermal control and environmental sensing/housekeeping including temperature, pressure, humidity, and acceleration. Electronics consist of a single Master PCB that interfaces to the spacecraft bus and regulates power and communication, plus LED, Detector, and Valve Manifold PCBs for each experiment. To facilitate ease of reuse on future missions, experiment electronics were designed to be compatible with a standard 3U small sat form factor and power bus, or to interface with a Master power/comm PCB for use in a larger satellite as in the case of PowerCell's flight on Eu:CROPIS.

biology↗

Scalable and automated CRISPR-based strain engineering using droplet microfluidics

Abstract We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system’s capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase ( galK ) in E. coli and (2) targeted engineering of the glutamine synthetase gene ( glnA ) and the blue-pigment synthetase gene ( bpsA ) to improve indigoidine production in E. coli .

42 ENGINEERING↗