Programmable Static Droplet Array for the Analysis of Cell-Cell Communication in a Confined Microenvironment.

Direct cell-cell communication can occur through various chemical and mechanical signals. However, available cell culture systems lack single-cell resolution and are often limited by sensitivity and accuracy. In this study, we present an accurate, efficient and controllable microfluidic device that can be used for in situ monitoring of natural cell-cell contact and signaling processes in a confined microenvironment. This innovative static droplet array (SDA) enables highly efficient trapping, encapsulation, arraying, storage, and incubation of defined cell populations. For proof-of-principle experiments, we monitored the response of budding yeast to peptide mating pheromones, as it is one of the best understood examples of eukaryotic cell-cell communication. Specifically, we measured the yeast response to varying concentration of synthetic MATα-type mating factor, as well as varying the cell number ratio of MATα and MATa in a confined space. We found clear morphological and doubling-time changes during the mating reaction with a significantly higher accuracy than conventional methods. Further, phenotypic analysis of data generated with the microfluidic static droplet array allowed distinguishing the function of genes in yeast mutants defective for different aspects of pheromone signaling. Taken together, the microfluidic platform provides a valuable research tool to study cell-cell communication and signaling in a controlled microenvironment with the sensitivity and accuracy required for screening and long-term phenotypic analysis.

Controlled fabrication of multicompartmental polymeric microparticles by sequential micromolding via surface-tension-induced droplet formation.

Polymeric multicompartmental microparticles have significant potential in many applications due to the capability to hold various functions in discrete domains within a single particle. Despite recent progress in microfluidic techniques, simple and scalable fabrication methods for multicompartmental particles remain challenging. This study reports a simple sequential micromolding method to produce monodisperse multicompartmental particles with precisely controllable size, shape, and compartmentalization. Specifically, our fabrication procedure involves sequential formation of primary and secondary compartments in micromolds via surface-tension-induced droplet formation coupled with simple photopolymerization. Results show that monodisperse bicompartmental particles with precisely controllable size, shape, and chemistry can be readily fabricated without sophisticated control or equipment. This technique is then extended to produce multicompartmental particles with controllable number of compartments and their size ratios through simple design of mold geometry. Also, core-shell particles with controlled number of cores for primary compartments can be readily produced by simple tuning of wettability. Finally, we demonstrate that the as-prepared multicompartmental particles can exhibit controlled release of multiple payloads based on design of particle compositions. Combined, these results illustrate a simple, robust, and scalable fabrication of highly monodisperse and complex multicompartmental particles in a controlled manner based on sequential micromolding.

A rapid one-step fabrication of patternable superhydrophobic surfaces driven by Marangoni instability.

We present a facile and inexpensive approach without any fluorinated chemistry to create superhydrophobic surface with exceptional liquid repellency, transportation of oil, selective capture of oil, optical bar code, and self-cleaning. Here we show experimentally that the control of evaporation is important and can be used to form superhydrophobic surface driven by Marangoni instability: the method involves in-situ photopolymerization in the presence of a volatile solvent and porous PDMS cover to afford superhydrophobic surfaces with the desired combination of micro- and nanoscale roughness. The porous PDMS cover significantly affects Marangoni convection of coating fluid, inducing composition gradients at the same time. In addition, the change of concentration of ethanol is able to produce versatile surfaces from hydrophilic to superhydrophobic and as a consequence to determine contact angles as well as roughness factors. In conclusion, the control of evaporation under the polymerization provides a convenient parameter to fabricate the superhydrophobic surface, without application of fluorinated chemistry and the elegant nanofabrication technique.

Synthetic cellular communication-based screening for strains with improved 3-hydroxypropionic acid secretion.

Although cellular secretion is important in industrial biotechnology, its assessment is difficult due to the lack of efficient analytical methods. This study describes a synthetic cellular communication-based microfluidic platform for screening strains with the improved secretion of 3-hydroxypropionic acid (3-HP), an industry-relevant platform chemical. 3-HP-secreting cells were compartmentalized in droplets, with receiving cells equipped with a genetic circuit that converts the 3-HP secretion level into an easily detectable signal. This platform was applied to identify Escherichia coli genes that enhance the secretion of 3-HP. As a result, two genes (setA, encoding a sugar exporter, and yjcO, encoding a Sel1 repeat-containing protein) found by this platform enhance the secretion of 3-HP and its production. Given the increasing design capability for chemical-detecting cells, this platform has considerable potential in identifying efflux pumps for not only 3-HP but also many important chemicals.

Quantitative analysis of yeast MAPK signaling networks and crosstalk using a microfluidic device.

Eukaryotic cells developed complex mitogen-activated protein kinase (MAPK) signaling networks to sense their intra- and extracellular environment and respond to various stress conditions. For example, S. cerevisiae uses five distinct MAP kinase pathways to orchestrate meiosis or respond to mating pheromones, osmolarity changes and cell wall stress. Although each MAPK module has been studied individually, the mechanisms underlying crosstalk between signaling pathways remain poorly understood, in part because suitable experimental systems to monitor cellular outputs when applying different signals are lacking. Here, we investigate the yeast MAPK signaling pathways and their crosstalk, taking advantage of a new microfluidic device coupled to quantitative microscopy. We designed specific micropads to trap yeast cells in a single focal plane, and modulate the magnitude of a given stress signal by microfluidic serial dilution while keeping other signaling inputs constant. This approach enabled us to quantify in single cells nuclear relocation of effectors responding to MAPK activation, like Yap1 for oxidative stress, and expression of stress-specific reporter expression, like pSTL1-qV and pFIG1-qV for high-osmolarity or mating pheromone signaling, respectively. Using this quantitative single-cell analysis, we confirmed bimodal behavior of gene expression in response to Hog1 activation, and quantified crosstalk between the pheromone- and cell wall integrity (CWI) signaling pathways. Importantly, we further observed that oxidative stress inhibits pheromone signaling. Mechanistically, this crosstalk is mediated by Pkc1-dependent phosphorylation of the scaffold protein Ste5 on serine 185, which prevents Ste5 recruitment to the plasma membrane.

Microfluidic preparation of monodisperse polymeric microspheres coated with silica nanoparticles.

The synthesis of organic-inorganic hybrid particles with highly controlled particle sizes in the micrometer range is a major challenge in many areas of research. Conventional methods are limited for nanometer-scale fabrication because of the difficulty in controlling the size. In this study, we present a microfluidic method for the preparation of organic-inorganic hybrid microparticles with poly (1,10-decanediol dimethacrylate-co-trimethoxysillyl propyl methacrylate) (P (DDMA-co-TPM)) as the core and silica nanoparticles as the shell. In this approach, the droplet-based microfluidic method combined with in situ photopolymerization produces highly monodisperse organic microparticles of P (DDMA-co-TPM) in a simple manner, and the silica nanoparticles gradually grow on the surface of the microparticles prepared via hydrolysis and condensation of tetraethoxysilane (TEOS) in a basic ammonium hydroxide medium without additional surface treatment. This approach leads to a reduction in the number of processes and allows drastically improved size uniformity compared to conventional methods. The morphology, composition, and structure of the hybrid microparticles are analyzed by SEM, TEM, FT-IR, EDS, and XPS, respectively. The results indicate the inorganic shell of the hybrid particles consists of SiO2 nanoparticles of approximately 60 nm. Finally, we experimentally describe the formation mechanism of a silica-coating layer on the organic surface of polymeric core particles.

A highly addressable static droplet array enabling digital control of a single droplet at pico-volume resolution.

Droplet-based microfluidics enabling exquisite liquid-handling has been developed for diagnosis, drug discovery and quantitative biology. Compartmentalization of samples into a large number of tiny droplets is a great approach to perform multiplex assays and to improve reliability and accuracy using a limited volume of samples. Despite significant advances in microfluidic technology, individual droplet handling in pico-volume resolution is still a challenge in obtaining more efficient and varying multiplex assays. We present a highly addressable static droplet array (SDA) enabling individual digital manipulation of a single droplet using a microvalve system. In a conventional single-layer microvalve system, the number of microvalves required is dictated by the number of operation objects; thus, individual trap-and-release on a large-scale 2D array format is highly challenging. By integrating double-layer microvalves, we achieve a "balloon" valve that preserves the pressure-on state under released pressure; this valve can allow the selective releasing and trapping of 7200 multiplexed pico-droplets using only 1 µL of sample without volume loss. This selectivity and addressability completely arranged only single-cell encapsulated droplets from a mixture of droplet compositions via repetitive selective trapping and releasing. Thus, it will be useful for efficient handling of miniscule volumes of rare or clinical samples in multiplex or combinatory assays, and the selective collection of samples.

Microfluidic preparation of a highly active and stable catalyst by high performance of encapsulation of polyvinylpyrrolidone (PVP)-Pt nanoparticles in microcapsules.

The encapsulation of active metals in microcapsules would be highly advantageous in maintaining or improving the reaction performance of an array of widely used chemical reactions. However, conventional methods suffer from low uniformity, complicated fabrication steps, sintering, leaching, decline of catalytic activity, and/or poor reusability. Here, we report an efficient microfluidic approach to encapsulate Pt nanoparticle stabilized by polyvinylpyrrolidone (PVP) in photocurable double-emulsion droplets with semipermeable thin shells. The encapsulated catalysts are prepared by the in situ photopolymerization of a double emulsion. The rapid and exquisite microfluidics-based fabrication process successfully generates monodisperse microcapsules without loss of the PVP-Pt nanoparticles, which is the first demonstration of the microfluidic encapsulation of active metal with promising catalytic activity. Specifically, compared to quasi-homogeneous catalysis of PVP-Pt nanoparticles for 4-nitrophenol hydrogenation, the encapsulated PVP-Pt nanoparticles demonstrate excellent catalytic activity, a leaching-proof nature, and high reusability under the same reaction conditions. We envision that the approach described here may be an example of elegant catalyst design to efficiently overcome difficult problems in active-metal encapsulation and to dramatically enhance catalytic activity by taking advantage of the unique aspects of microfluidic methods.

On-chip analysis, indexing and screening for chemical producing bacteria in a microfluidic static droplet array.

Economic production of chemicals from microbes necessitates development of high-producing strains and an efficient screening technology is crucial to maximize the effect of the most popular strain improvement method, the combinatorial approach. However, high-throughput screening has been limited for assessment of diverse intracellular metabolites at the single-cell level. Herein, we established a screening platform that couples a microfluidic static droplet array (SDA) and an artificial riboswitch to analyse intracellular metabolite concentration from single microbial cells. Using this system, we entrapped single Escherichia coli cells in SDA to measure intracellular l-tryptophan concentrations. It was validated that intracellular l-tryptophan concentration can be evaluated by the fluorescence from the riboswitch. Moreover, high-producing strains were successfully screened from a mutagenized library, exhibiting up to 145% productivity compared to its parental strain. This platform will be widely applicable to strain improvement for diverse metabolites by developing new artificial riboswitches.

Monitoring of chromosome dynamics of single yeast cells in a microfluidic platform with aperture cell traps.

Chromosome movement plays important roles in DNA replication, repair, genetic recombination, and epigenetic phenomena during mitosis and meiosis. In particular, chromosome movement in the nuclear space is essential for the reorganization of the nucleus. However, conventional methods for analyzing the chromosome movements in vivo have been limited by technical constraints of cell trapping, cell cultivation, oxygenation, and in situ imaging. Here, we present a simple microfluidic platform with aperture-based cell trapping arrays to monitor the chromosome dynamics in single living cells for a desired period of time. Under the optimized conditions, our microfluidic platform shows a single-cell trapping efficiency of 57%. This microfluidic approach enables in situ imaging of intracellular dynamics in living cells responding to variable input stimuli under the well-controlled microenvironment. As a validation of this microfluidic platform, we investigate the fundamental features of the dynamic cellular response of the individual cells treated with different stimuli and drug. We prove the basis for dynamic chromosome movement in single yeast cells to be the telomere and nuclear envelope ensembles that attach to and move in concert with nuclear actin cables. Therefore, these results illustrate the monitoring of cellular functions and obtaining of dynamic information at a high spatiotemporal resolution through the integration of a simple microfluidic platform.

Microfluidic static droplet array for analyzing microbial communication on a population gradient.

Quorum sensing (QS) is a type of cell-cell communication using signal molecules that are released and detected by cells, which respond to changes in their population density. A few studies explain that QS may operate in a density-dependent manner; however, due to experimental challenges, this fundamental hypothesis has never been investigated. Here, we present a microfluidic static droplet array (SDA) that combines a droplet generator with hydrodynamic traps to independently generate a bacterial population gradient into a parallel series of droplets under complete chemical and physical isolation. The SDA independently manipulates both a chemical concentration gradient and a bacterial population density. In addition, the bacterial population gradient in the SDA can be tuned by a simple change in the number of sample plug loading. Finally, the method allows the direct analysis of complicated biological events in an addressable droplet to enable the characterization of bacterial communication in response to the ratio of two microbial populations, including two genetically engineered QS circuits, such as the signal sender for acyl-homoserine lactone (AHL) production and the signal receiver bacteria for green fluorescent protein (GFP) expression induced by AHL. For the first time, we found that the population ratio of the signal sender and receiver indicates a significant and potentially interesting partnership between microbial communities. Therefore, we envision that this simple SDA could be a useful platform in various research fields, including analytical chemistry, combinatorial chemistry, synthetic biology, microbiology, and molecular biology.

A programmable microfluidic static droplet array for droplet generation, transportation, fusion, storage, and retrieval.

We present a programmable microfluidic static droplet array (SDA) device that can perform user-defined multistep combinatorial protocols. It combines the passive storage of aqueous droplets without any external control with integrated microvalves for discrete sample dispensing and dispersion-free unit operation. The addressable picoliter-volume reaction is systematically achieved by consecutively merging programmable sequences of reagent droplets. The SDA device is remarkably reusable and able to perform identical enzyme kinetic experiments at least 30 times via automated cross-contamination-free removal of droplets from individual hydrodynamic traps. Taking all these features together, this programmable and reusable universal SDA device will be a general microfluidic platform that can be reprogrammed for multiple applications.