Robust Microfluidic Platform Research Articles

Fluidic Interface for Surface-based DNA Origami Studies.

Traditionally, the use of DNA origami nanostructures (DONs) to study early cell signaling processes has been conducted using standard laboratory equipment with DONs typically utilized in solution. Surface-based technologies simplify the microscopic analysis of cells treated with DON agents by anchoring them to solid substrates, thus avoiding the complications of receptor-mediated endocytosis. A robust microfluidic platform for real-time monitoring and precise functionalization of surfaces with DONs was developed here. The combination of controlled flow conditions with an upright total internal reflection fluorescence microscope enabled the kinetic analysis of the immobilization of DONs on DNA-functionalized surfaces. The results revealed that DON morphology and binding tags influence the binding kinetics and that DON hybridization on surfaces is more effective in microfluidic devices with larger-than-standard dimensions, addressing the low diffusivity challenge of DONs. The platform enabled the decoration of DONs with protein-binding ligands and in situ investigation of ligand occupancy on DONs to produce high-quality bioactive surfaces. These surfaces were used to recruit and activate the epidermal growth factor receptor (EGFR) through clustering in the membranes of living cancer cells (MCF-7) using an antagonistic antibody (Panitumumab). The activation was quantified depending on the interligand distances of the EGFR-targeting antibody.

Extracellular vesicle and lipoprotein diagnostics (ExoLP-Dx) with membrane sensor: A robust microfluidic platform to overcome heterogeneity.

The physiological origins and functions of extracellular vesicles (EVs) and lipoproteins (LPs) propel advancements in precision medicine by offering non-invasive diagnostic and therapeutic prospects for cancers, cardiovascular, and neurodegenerative diseases. However, EV/LP diagnostics (ExoLP-Dx) face considerable challenges. Their intrinsic heterogeneity, spanning biogenesis pathways, surface protein composition, and concentration metrics complicate traditional diagnostic approaches. Commonly used methods such as nanoparticle tracking analysis, enzyme-linked immunosorbent assay, and nuclear magnetic resonance do not provide any information about their proteomic subfractions, including active proteins/enzymes involved in essential pathways/functions. Size constraints limit the efficacy of flow cytometry for small EVs and LPs, while ultracentrifugation isolation is hampered by co-elution with non-target entities. In this perspective, we propose a charge-based electrokinetic membrane sensor, with silica nanoparticle reporters providing salient features, that can overcome the interference, long incubation time, sensitivity, and normalization issues of ExoLP-Dx from raw plasma without needing sample pretreatment/isolation. A universal EV/LP standard curve is obtained despite their heterogeneities.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.

Self-organized dynamics and the transition to turbulence of confined active nematics

We study how confinement transforms the chaotic dynamics of bulk microtubule-based active nematics into regular spatiotemporal patterns. For weak confinements in disks, multiple continuously nucleating and annihilating topological defects self-organize into persistent circular flows of either handedness. Increasing confinement strength leads to the emergence of distinct dynamics, in which the slow periodic nucleation of topological defects at the boundary is superimposed onto a fast procession of a pair of defects. A defect pair migrates toward the confinement core over multiple rotation cycles, while the associated nematic director field evolves from a distinct double spiral toward a nearly circularly symmetric configuration. The collapse of the defect orbits is punctuated by another boundary-localized nucleation event, that sets up long-term doubly periodic dynamics. Comparing experimental data to a theoretical model of an active nematic reveals that theory captures the fast procession of a pair of [Formula: see text] defects, but not the slow spiral transformation nor the periodic nucleation of defect pairs. Theory also fails to predict the emergence of circular flows in the weak confinement regime. The developed confinement methods are generalized to more complex geometries, providing a robust microfluidic platform for rationally engineering 2D autonomous flows.

Dynamic Halbach array magnet integrated microfluidic system for the continuous-flow separation of rare tumor cells.

Circulating tumor cells (CTCs), the most representative rare cells in peripheral blood, have received great attention due to their clinical utility in liquid biopsy. The downstream analysis of intact CTCs isolated from peripheral blood provides important clinical information for personalized medicine. However, current CTC isolation and detection methods have been challenged by their extreme rarity and heterogeneity. In this study, we developed a novel microfluidic system with a continuously moving Halbach array magnet (dHAMI microfluidic system) for negative isolation CTCs from whole blood, which aimed to capture non-target white blood cells (WBCs) and elute target CTCs. The dynamic and continuous movement of the Halbach array magnet generated a continuous magnetic force acting on the magnetic bead-labelled WBCs in the continuous-flow fluid to negatively exclude the WBCs from the CTCs. Furthermore, the continuously moving magnetic field effectively eliminated the effect of magnetic bead aggregation on the fluid flow to realize the continuous-flow separation of the CTCs without a sample loading volume limitation. The experimental procedure for CTC negative isolation using the dHAMI microfluidic system could be completed within 40 min. Under the optimized experimental conditions of the dHAMI microfluidic system, including the flow rate and concentration of the immunomagnetic bead, the average CTC capture rate over a range of spiked cell numbers (50–1000 cancer cells per mL) was up to 91.6% at a flow rate of 100 μL min−1. Finally, the CTCs were successfully detected in 10 of 10 (100%) blood samples from patients with cancer. Therefore, the dHAMI microfluidic system could effectively isolate intact and heterogeneous CTCs for downstream cellular and molecular analyses, and this robust microfluidic platform with an excellent magnetic manipulation performance also has great application potential for the separation of other rare cells.

Controlled fabrication of multi-core alginate microcapsules

In this work, we present a robust microfluidic platform for controlled and complete on-chip generation of alginate microcapsules with single and double liquid cores. A combined Coflow and T-junction configuration implemented in a hybrid glass-PDMS (Polydimethylsiloxane) device is used for the generation of microcapsules with oil as liquid core. Frequency matching of oil-alginate double emulsion generation with that of aqueous Calcium chloride droplet generation allows for controlled merging of the two, resulting in reliable production of microcapsules. Confocal imaging of microcapsule cross-section reveals presence of intact liquid core. In the case of double core microcapsules, the two cores are well separated by alginate layer ensuring their long term stability. The current approach is expected to have advantages over existing techniques for liquid core microcapsule generation in terms of continuity of the process, control over core stability, and non-damage to cells when used for cell encapsulation applications.

Characterization of three-dimensional cancer cell migration in mixed collagen-Matrigel scaffolds using microfluidics and image analysis.

Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions.Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix.In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs.

A versatile microfluidic device for high throughput production of microparticles and cell microencapsulation.

Biocompatible microparticles are valuable tools in biomedical research for applications such as drug delivery, cell transplantation therapy, and analytical assays. However, their translation into clinical research and the pharmaceutical industry has been slow due to the lack of techniques that can produce microparticles with controlled physicochemical properties at high throughput. We introduce a robust microfluidic platform for the production of relatively homogeneous microdroplets at a generation frequency of up to 3.1 MHz, which is about three orders of magnitude higher than the production rate of a conventional microfluidic drop maker. We demonstrated the successful implementation of our device for production of biocompatible microparticles with various crosslinking mechanisms and cell microencapsulation with high cell viability.

Oxygen Tension and Riboflavin Gradients Cooperatively Regulate the Migration of Shewanella oneidensis MR-1 Revealed by a Hydrogel-Based Microfluidic Device

Shewanella oneidensis is a model bacterial strain for studies of bioelectrochemical systems (BESs). It has two extracellular electron transfer pathways: (1) shuttling electrons via an excreted mediator riboflavin; and (2) direct contact between the c-type cytochromes at the cell membrane and the electrode. Despite the extensive use of S. oneidensis in BESs such as microbial fuel cells and biosensors, many basic microbiology questions about S. oneidensis in the context of BES remain unanswered. Here, we present studies of motility and chemotaxis of S. oneidensis under well controlled concentration gradients of two electron acceptors, oxygen and oxidized form of riboflavin (flavin+), using a newly developed microfluidic platform. Experimental results demonstrate that either oxygen or flavin+ is a chemoattractant to S. oneidensis. The chemotactic tendency of S. oneidensis in a flavin+ concentration gradient is significantly enhanced in an anaerobic in contrast to an aerobic condition. Furthermore, either a low oxygen tension or a high flavin+ concentration considerably enhances the speed of S. oneidensis. This work presents a robust microfluidic platform for generating oxygen and/or flavin+ gradients in an aqueous environment, and demonstrates that two important electron acceptors, oxygen and oxidized riboflavin, cooperatively regulate S. oneidensis migration patterns. The microfluidic tools presented as well as the knowledge gained in this work can be used to guide the future design of BESs for efficient electron production.

Microfluidics: A Versatile and Robust Microfluidic Platform Toward High Throughput Synthesis of Homogeneous Nanoparticles with Tunable Properties (Adv. Mater. 14/2015)

Microfluidics: A Versatile and Robust Microfluidic Platform Toward High Throughput Synthesis of Homogeneous Nanoparticles with Tunable Properties (Adv. Mater. 14/2015)

A versatile and robust microfluidic platform toward high throughput synthesis of homogeneous nanoparticles with tunable properties.

A versatile and robust microfluidic nanoprecipitation platform for high throughput synthesis of nanoparticles is fabricated. The versatility of this platform is proven through the successful preparation of different types of nanoparticles. This platform presents great robustness, with homogeneous nanoparticles always being obtained, regardless of the formulation parameters. The diameter and surface charge of the prepared nanoparticles can also be easily tuned.

Microfluidic platforms with monolithically integrated hierarchical apertures for the facile and rapid formation of cargo-carrying vesicles.

We fabricated a simple yet robust microfluidic platform with monolithically integrated hierarchical apertures. This platform showed efficient diffusive mixing of the introduced lipids through approximately 8000 divisions with tiny pores (~5 μm in diameter), resulting in massive, real-time production of various cargo-carrying particles via multi-hydrodynamic focusing.

Microfluidic chemical cytometry of peptide degradation in single drug-treated acute myeloid leukemia cells.

Microfluidic systems show great promise for single-cell analysis; however, as these technologies mature, their utility must be validated by studies of biologically relevant processes. An important biomedical application of these systems is characterization of tumor cell heterogeneity. In this work, we used a robust microfluidic platform to explore the heterogeneity of enzyme activity in single cells treated with a chemotherapeutic drug. Using chemical cytometry, we measured peptide degradation in the U937 acute myeloid leukemia (AML) cell line in the presence and absence of the aminopeptidase inhibitor Tosedostat (CHR-2797). The analysis of 99 untreated cells revealed rapid and consistent degradation of the peptide reporter within 20 min of loading. Results from drug-treated cells showed inhibited, but ongoing degradation of the reporter. Because the device operates at an average sustained throughput of 37 ± 7 cells/h, we were able to sample cells over the course of this time-dependent degradation. In data from 498 individual drug-treated cells, we found a linear dependence of degradation rate on amount of substrate loaded superimposed upon substantial heterogeneity in peptide processing in response to inhibitor treatment. Importantly, these data demonstrated the potential of microfluidic systems to sample biologically relevant analytes and time-dependent processes in large numbers of single cells.

Concentration gradient generation of multiple chemicals using spatially controlled self-assembly of particles in microchannels

We present a robust microfluidic platform for the stable generation of multiple chemical gradients simultaneously using in situ self-assembly of particles in microchannels. This proposed device enables us to generate stable and reproducible diffusion-based gradients rapidly without convection flow: gradients are stabilized within 5 min and are maintained steady for several hours. Using this device, we demonstrate the dynamic position control of bacteria by introducing the sequential directional change of chemical gradients. Green Fluorescent Protein (GFP)-expressing bacterial cells, allowing quantitative monitoring, show not only tracking motion according to the directional control of chemical gradients, but also the gradual loss of sensitivity when exposed to the sequential attractants because of receptor saturation. In addition, the proposed system can be used to study the preferential chemotaxis assay of bacteria toward multiple chemical sources, since it is possible to produce multiple chemical gradients in the main chamber; aspartate induces the most preferential chemotaxis over galactose and ribose. The microfluidic device can be easily fabricated with a simple and cost effective process based on capillary pressure and evaporation for particle assembly. The assembled particles create uniform porous membranes in microchannels and its porosity can be easily controlled with different size particles. Moreover, the membrane is biocompatible and more robust than hydrogel-based porous membranes. The proposed system is expected to be a useful tool for the characterization of bacterial responses to various chemical sources, screening of bacterial cells, synthetic biology and understanding many cellular activities.

Microbridge structures for uniform interval control of flowing droplets in microfluidic networks

Precise temporal control of microfluidic droplets such as synchronization and combinatorial pairing of droplets is required to achieve a variety range of chemical and biochemical reactions inside microfluidic networks. Here, we present a facile and robust microfluidic platform enabling uniform interval control of flowing droplets for the precise temporal synchronization and pairing of picoliter droplets with a reagent. By incorporating microbridge structures interconnecting the droplet-carrying channel and the flow control channel, a fluidic pressure drop was derived between the two fluidic channels via the microbridge structures, reordering flowing droplets with a defined uniform interval. Through the adjustment of the control oil flow rate, the droplet intervals were flexibly and precisely adjustable. With this mechanism of droplet spacing, the gelation of the alginate droplets as well as control of the droplet interval was simultaneously achieved by additional control oil flow including calcified oleic acid. In addition, by parallel linking identical microfluidic modules with distinct sample inlet, controlled synchronization and pairing of two distinct droplets were demonstrated. This method is applicable to facilitate and develop many droplet-based microfluidic applications, including biological assay, combinatorial synthesis, and high-throughput screening.

High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging

Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 µm diameter, 8 µm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 µm diameter and 8 µm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 µM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.

Editorial: A brief welcome to this new electronic journal on microfluidic applications to biology and medicine

Spurred by breakthroughs in molecular characterization and microcircuit fabrication techniques, the last decade has seen significant growth in the fields of biophysics and biomedical engineering. Quantum dots, PCR amplification, optical tweezers, AFM, nanocolloids, electrosprays, etc., have catalyzed a myriad of new ways to detect antigens, assay proteins, diagnose diseases, and decipher the genome. Nanomedicine based on immunocolloids is no longer a dream. The time is ripe to bring these diagnostic and drug delivery techniques out of the laboratory and into miniature chips that can be used in the field. Glucose detection kits, DNA hybridization microarrays for genetic identification, insulin inhalers, etc., are just the first of such products that can drive a major biotechnology industry in the near future. This extension from the lab to field requires, however, a robust microfluidic platform that is the focus of our new journal, Biomicrofluidics. These microfluidic techniques allow us to transport fluids and manipulate colloid∕cells∕molecules in small devices. Cellular and molecular level operations, like electroporation and chip-scale optical spectroscopy of molecules, all require specific microfluidic designs that are not yet available. Indeed, the same microfluidic know-how can be used to assemble biological materials (membranes and bones) via directed assembly. The large Editorial Board of Biomicrofluidics is interdisciplinary and multinational, which reflects the composition of this new community. With rapid turnaround and wide circulation, we hope to promote interaction among the large but scattered research communities over the globe. The open-access format and full-media capability are designed to allow rapid dissemination of new techniques and information to the biotechnology industry. The Blog page on the journal website is intended to be a bulletin and sounding board for the community to announce conferences and to solicit responses to new ideas∕technological challenges. We anticipate a major growth in the biotechnology industry and a corresponding increase in microfluidic research. It is our hope that Biomicrofluidics will serve this growing community by offering a common link and a communication conduit among the various scientific and engineering disciplines that participate in this exciting new field. We welcome your submissions; visit the journal’s submission and peer review website at http://bmf.peerx-press.org.

Polar stimulation and constrained cell migration in microfluidic channels

Asymmetrical delivery of stimuli to moving cells for perturbing spatially-heterogeneous intracellular signaling is an experimental challenge not adequately met by existing technologies. Here, we report a robust microfluidic platform allowing localized treatment of the front and/or back of moving cells which crawl through narrow channels that they completely occlude. The enabling technical element for this study is a novel design for precise, passive balancing of flow inside the microfluidic device by contacting two fluid streams before splitting them again. The microchannels constrain cell morphology and induce qualitative and quantitative changes in neutrophil chemotaxis that mimic cells crawling through tissues.