Production of uniform droplets using membrane, microchannel and microfluidic emulsification devices

This review provides an overview of major microengineering emulsification techniques for production of monodispersed droplets. The main emphasis has been put on membrane emulsification using Shirasu Porous Glass and microsieve membrane, microchannel emulsification using grooved-type and straight-through microchannel plates, microfluidic junctions and flow focusing microfluidic devices. Microfabrication methods for production of planar and 3D poly(dimethylsiloxane) devices, glass capillary microfluidic devices and single-crystal silicon microchannel array devices have been described including soft lithography, glass capillary pulling and microforging, hot embossing, anisotropic wet etching and deep reactive ion etching. In addition, fabrication methods for SPG and microseive membranes have been outlined, such as spinodal decomposition, reactive ion etching and ultraviolet LIGA (Lithography, Electroplating, and Moulding) process. The most widespread application of micromachined emulsification devices is in the synthesis of monodispersed particles and vesicles, such as polymeric particles, microgels, solid lipid particles, Janus particles, and functional vesicles (liposomes, polymersomes and colloidosomes). Glass capillary microfluidic devices are very suitable for production of core/shell drops of controllable shell thickness and multiple emulsions containing a controlled number of inner droplets and/or inner droplets of two or more distinct phases. Microchannel emulsification is a very promising technique for production of monodispersed droplets with droplet throughputs of up to 100 l h−1

Single-cell droplet microfluidics for metagenomics and cancer multiomics

Cellular heterogeneity is inherent to many biological systems, across both normal and disease states. For example, diverse ensembles of microbes in the natural environment fulfill distinct roles related to nutrient metabolism and gas fixation. In human cancers, genetic and phenotypic heterogeneity is observed among cells originating from a common oncogenic clone. Understanding biological heterogeneity, whether for metabolic engineering applications or the design of cancer therapeutics, begins at the fundamental unit of the organism: a single cell. Droplet microfluidics enables analyses of single cells at a biologically-relevant scale through rapid compartmentalization and manipulation of millions of parallel reactions. In this thesis, I describe the development and application of single-cell genomics platforms leveraging droplet microfluidics to interrogate many individual genomes. These technologies enable single-cell metagenomics and multiomic analysis of single cancer cells, providing new insights into the extent of cellular heterogeneity and its implications across biology

A general strategy for expanding polymerase function by droplet microfluidics

abstract: Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson–Crick base pairing.The final version of this article, as published in Nature Communications, can be viewed online at: https://www.nature.com/articles/ncomms1123

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

© 2017, National Academy of Sciences. All rights reserved.Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity

Production of uniform droplets using membrane, microchannel and microfluidic emulsification devices

This paper deals with the political nature of mafias that protect drug trafficking from a broadly mentioned but poorly studied aspect in the same concept of mafia, namely, the articulation of interests of diverse social groups within their protection offer. Both types of mafias, the ones that enjoy social control and the ones that do not enjoy major social interaction, aims to impose decisions favorable to their interests. The big difference is that decisions of the former ones tend to consider the social structure transformed by drug trafficking. Any attempt to repress these mafias means now an attack to the structural interests of diverse social groups. A specific case is used to illustrate this political approach on mafia: the war of Pablo Escobar against the Colombian state.Este artículo es una aproximación al carácter político de las mafias que protegen el tráfico de drogas desde una perspectiva mencionada pero poco tratada dentro del concepto mismo de mafia: la articulación de intereses de amplios grupos sociales dentro de su oferta de protección. Tanto las mafias de la droga que gozan de dominación social como las que no tienen mayor interacción social toman decisiones dirigidas a la imposición de sus intereses. La gran diferencia está en que las decisiones de las primeras tienden a involucrar la estructura social que ha sido transformada por el narcotráfico. Cualquier intento por reprimir a las mafias involucra ahora un ataque a los intereses estructurales de amplios sectores sociales. Un caso concreto servirá para ilustrar esta lectura política del narcotráfico: la guerra de Pablo Escobar contra el estado

Development of a Microfluidics-Based Screening Assay for the High-Throughput Directed Evolution of Artificial Metalloenzymes

The present PhD thesis summarizes the scientific work conducted in the research group of Prof. Dr. Thomas R. Ward during the years 2016–2021. Research in the Ward group is focused on the development and optimization of artificial metalloenzymes with non-natural activities. These hybrid catalysts, resulting from an incorporation of a metal–containing cofactor within a protein or DNA scaffold, and can be optimized by either chemical or genetic means. The main part of this thesis deals with the genetic optimization of such systems and the development of higher throughput screening assays to facilitate the process. First attempts dealt with the development of a selection-based assay relying on the Carroll rearrangement (Chapter 2.6). Following, more high-throughput assays such as screening of cells relying on a fluorescent reporter protein (Chapter 3) or the screening of activity by an agar plate screening assay were pursued (Chapter 4.2). The main part of the thesis focuses on the method development of an ultrahigh-throughput screening platform for the in vivo directed evolution of artificial metalloenzymes using droplet microfluidics. The combination of ArMs and droplet microfluidics, can be a powerful tool for propelling directed evolution-based research forward. Systematic and high-throughput screening of ArMs in vivo using double emulsions could allow the screening of a much bigger sequence space, which is, to date, challenging. Identifying cooperative effects to improve catalysis or even remodelling whole enzymes to achieve new-to-nature reactivities are only two potential examples. Reactions based on ArMs could ultimately provide aqueous, environmentally friendly reaction pathways for industrial applications. Additionally, such big data sets could also be used as an input for machine learning applications, to further study active site plasticity, reaction pathways, or even protein-folding mechanisms. The developed method was then applied to libraries of different types and sizes, and recent findings of these screenings are highlighted in the fourth chapter. During the time in the research group of Prof. Dr. Ward, a deeper knowledge in molecular biology, especially library design, high-throughput screening using different approaches, microfluidic method development and fluorescence activated cell sorting (FACS), and the use of different sequencing techniques was garnered

Application of Flow Cytometry as Novel Technology in Studying Lipid Oxidation in Oil-in-Water Emulsions

The body of literature on the impact of emulsion particle size on oxidation rates is unclear. This could be because emulsions are typically polydisperse and the oxidation rate of individual droplets is impossible to discern. Flow cytometry is a technique for studying individual cells and their subpopulations using fluorescence technologies. It is possible that individual emulsion droplets could also be characterized by flow cytometry as a novel approach for studying lipid oxidation. Typical emulsion droplets are too small to be visualized by flow cytometer, so emulsions were prepared to have droplets \u3e 2 μm; weighting agent and xanthan gum were added to minimize creaming during storage. A radical-sensitive lipid-soluble fluorescence probe (BODIPY665/676) was added to the lipid used to prepare the emulsion so that the susceptibility of individual emulsion droplets could be determined. The results showed that in a polydisperse emulsion system, small droplets were oxidized faster than large droplets. Using mixtures of emulsions with and without prooxidants, it was possible to see the transfer of prooxidants between droplets, a process that is influenced by surfactant and salt concentrations. For example, surfactants micelles can transfer prooxidants to neighboring non-oxidized droplets and cause fluorescence loss when surfactant concentration was higher than critical micelle concentration (CMC). Transfer of prooxidants was promoted by adding NaCl and free fatty acid which could be attributed to the lower CMC. This study showed the potential for applying flow cytometry on oxidation of individual emulsion droplets

Droplet Microfluidics

Droplet microfluidics has dramatically developed in the past decade and has been established as a microfluidic technology that can translate into commercial products. Its rapid development and adoption have relied not only on an efficient stabilizing system (oil and surfactant), but also on a library of modules that can manipulate droplets at a high-throughput. Droplet microfluidics is a vibrant field that keeps evolving, with advances that span technology development and applications. Recent examples include innovative methods to generate droplets, to perform single-cell encapsulation, magnetic extraction, or sorting at an even higher throughput. The trend consists of improving parameters such as robustness, throughput, or ease of use. These developments rely on a firm understanding of the physics and chemistry involved in hydrodynamic flow at a small scale. Finally, droplet microfluidics has played a pivotal role in biological applications, such as single-cell genomics or high-throughput microbial screening, and chemical applications. This Special Issue will showcase all aspects of the exciting field of droplet microfluidics, including, but not limited to, technology development, applications, and open-source systems

Combinatorial microfluidic droplet engineering for biomimetic material synthesis

Although droplet-based systems are used in a wide range of technologies, opportunities for systematically customizing their interface chemistries remain relatively unexplored. This article describes a new microfluidic strategy for rapidly tailoring emulsion droplet compositions and properties. The approach utilizes a simple platform for screening arrays of droplet-based microfluidic devices and couples this with combinatorial selection of the droplet compositions. Through the application of genetic algorithms over multiple screening rounds, droplets with target properties can be rapidly generated. The potential of this method is demonstrated by creating droplets with enhanced stability, where this is achieved by selecting carrier fluid chemistries that promote titanium dioxide formation at the droplet interfaces. The interface is a mixture of amorphous and crystalline phases, and the resulting composite droplets are biocompatible, supporting in vitro protein expression in their interiors. This general strategy will find widespread application in advancing emulsion properties for use in chemistry, biology, materials and medicine

Recent developments in manufacturing micro- and nano-particles from emulsion droplets

Membrane and microfluidic devices are relatively new tools for highly controlled production of particles. This review focuses on the recent developments in this area, ranging from the production of simple and double emulsions of different types and morphologies (e.g. multiple core-shell structures, outer drops with controlled number of internal droplets, etc) to highly sophisticated functional products such as polymerosomes, asymmetric lipid vesicles, and core-shell particles. Other emerging technologies that extend the capabilities into different membrane materials and operation methods (such as rotating stainless steel membrane with laser drilled pores) and manufacturing approaches (extrusion of pure to-be-dispersed phase or coarsely emulsified feeds) are introduced. The use of microfluidic T-junctions, flow focusing devices and silicon microchannel array devices is also reviewed. The results of experimental work carried out by cited researchers in the field together with those of the current authors are presented in a tabular form in a rigorous and systematic manner. These demonstrate a wide range of products that can be manufactured from emulsions using different solidification techniques