RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.

Synthetic biology and metabolic engineering seek to re-engineer microbes into living foundries for the production of high value chemicals. Through a design-build-test cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production

Combinatorial drug discovery in nanoliter droplets

Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here, we report a high-throughput system for nanoliter-scale phenotypic screening that formulates a chemical library in nanoliter droplet emulsions and automates the construction of chemical combinations en masse using parallel droplet processing. We applied this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics against Escherichia coli, a model gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against gram-positive bacteria but are not effective by themselves to resolve gram-negative infections. Keywords: high-throughput screening; nanoliter droplet; drug synergy; antibiotics; small molecule

Macroporous materials: microfluidic fabrication, functionalization and applications

This article provides an up-to-date highly comprehensive overview (594 references) on the state of the art of the synthesis and design of macroporous materials using microfluidics and their applications in different fields

Hydrogel microparticles for biosensing

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field. Keywords: Hydrogel; Biosensor; Microparticle; Multiplex assayNovartis Institutes of Biomedical Research (Presidential Fellowship)Novartis Institutes of Biomedical Research (Education Office)National Cancer Institute (U.S.) (Grant 5R21CA177393-02)National Science Foundation (U.S.) (Grant CMMI-1120724)Institute for Collaborative Biotechnologies (Grant W911NF-09-0001)United States. Army Research Offic

Single cell measurement of telomerase expression and splicing using microfluidic emulsion cultures.

Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β- variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response

Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex productio

Microchips and their significance in isolation of circulating tumor cells and monitoring of cancers

In micro-fluid systems, fluids are injected into extremely narrow polymer channels in small amounts such as micro-, nano-, or pico-liter scales. These channels themselves are embedded on tiny chips. Various specialized structures in the chips including pumps, valves, and channels allow the chips to accept different types of fluids to be entered the channel and along with flowing through the channels, exert their effects in the framework of different reactions. The chips are generally crystal, silicon, or elastomer in texture. These highly organized structures are equipped with discharging channels through which products as well as wastes of the reactions are secreted out. A particular advantage regarding the use of fluids in micro-scales over macro-scales lies in the fact that these fluids are much better processed in the chips when they applied as micro-scales. When the laboratory is miniaturized as a microchip and solutions are injected on a micro-scale, this combination makes a specialized construction referred to as "lab-on-chip". Taken together, micro-fluids are among the novel technologies which further than declining the costs; enhancing the test repeatability, sensitivity, accuracy, and speed; are emerged as widespread technology in laboratory diagnosis. They can be utilized for monitoring a wide spectrum of biological disorders including different types of cancers. When these microchips are used for cancer monitoring, circulatory tumor cells play a fundamental role

Sistemas microfuidicos de gotas para incorporaçao de acidos nucleicos em lipossomas cationicos e para transfecçao in vitro de células de mamiferos

Orientadores: Lucimara Gaziola de la Torre, Charles BaroudTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química, École Polytechnique - FrançaResumo: Este trabalho visou o uso de um sistema microfluídico de gotas para incorporar ácidos nucleicos em lipossomas catiônicos e outro para estudar o processo de transfecção em células de mamíferos. A primeira etapa do projeto utilizou um microdispositivo para incorporar pDNA em lipossomas catiônicos de modo a obter lipoplexos reprodutíveis e adequados para transfectar células dendríticas (DCs). Com esta finalidade, alguns parâmetros experimentais foram investigados, tais como vazões de entrada, manutenção das propriedades dos lipossomas após processamento no microdispositivo, características dos lipoplexos (tamanho, polidispersidade e carga) em função da razão molar de carga (R+/-) e do desenho do microdispositivo. Lipoplexos produzidos em microdispositivo com canal de serpentina largo e região de divisão de gotas que diminuem a polidispersidade dos lipoplexos, operando à razão de vazão água/óleo 0,25 e R+/- 1,5; 3; 5; 7 e 10 foram utilizados para transfectar DCs in vitro. Todos os lipoplexos foram capazes de transfectar as DCs e ao mesmo tempo proporciaonar a ativação das células. A segunda etapa do trabalho utilizou uma plataforma microfluídica de célula única para investigar e controlar as condições de transfecção, tendo em vista a otimização dos rendimentos de produção de proteínas recombinantes. Neste contexto, as células de ovário de hamster Chinês (CHO-S) foram transfectadas no microdispositivo com diferentes tipos de lipoplexos (R+/- 1,5; 3; 5) e monitoradas em relação à produção de proteína verde fluorescente (GFP) e viabilidade celular. A plataforma de célula única permite avaliar a heterogeneidade celular, revelando a presença de uma subpopulação que produz níveis elevados de GFP. Essas células com alta produção de GFP (HP) mostraram um aumento do tamanho celular em comparação à média da população. Além disso, a carga dos lipoplexes apresenta um importante papel na transfecção das células CHO-S, visto que os únicos lipoplexos com carga positiva R+/- 5 produziram mais HPs. A quantidade de pDNA entregue às células afeta a produção de proteína, já que os lipoplexos com mais pDNA R+/- 1,5 aumentaram a produtividade específica de GFP das HPs. Esta tese foi desenvolvida no âmbito de um programa de co-tutela entre a Universidade Estadual de Campinas, Brasil e a École Polytechnique, França. Em geral, este trabalho apresenta contribuições originais para as áreas da microfluídica e da entrega de genesAbstract: This work aimed at using one droplet-based microfluidic systems to incorporate nucleic acids into cationic liposomes and another one to study the mammalian cell transfection process. In the first part of this study we used a droplet-based microfluidic system to complex cationic liposomes with pDNA in order to obtain reproducible and suitable lipoplexes to dendritic cells (DCs) transfection. For this purpose, some experimental parameters were investigated, such as inlet flow rates, the maintenance of liposomes¿ properties after microfluidic processing, lipoplex characteristics (size, polydispersity and zeta potential) as function of molar charge ratio (R+/-) and microchip design. Lipoplexes produced in a microchip with large serpentine channel and split region, which decreases lipoplex polydispersity, operating at ratio aqueous/oil flow rate 0.25 and R+/- 1.5, 3, 5, 7 and 10 were used to transfect DCs in vitro. All lipoplexes transfected DCs and resulted in cell activation. In the second part of this study we used a single-cell microfluidic platform to investigate and control transfection conditions, in view of optimizing the recombinant protein production by transfected cells. Chinese hamster ovary cells (CHO-S) were transfected in microchip with different types of lipoplexes (R+/- 1.5, 3, 5) and monitored by green fluorescent protein (GFP) production and cell viability. The single-cell platform enables to assess the heterogeneities of CHO-S population, revealing the presence of a subpopulation producing significantly high levels of GFP. These high producers (HP) showed increased cell size in comparison to the average population. Moreover, the charge of lipoplexes shows an important role to transfect CHO-S, since the unique positive charged lipoplex R+/- 5 produced more HPs. Additionally, the amount of pDNA delivered affects protein production, since R+/- 1.5 with more pDNA increased GFP specific productivity of HPs. This thesis was developed under the joint graduate program of the University of Campinas, Brazil and École Polytechnique, France. In general, this work presents original contributions in the areas of microfluidics and gene deliveryDoutoradoDesenvolvimento de Processos BiotecnologicosDoutora em Engenharia Quimica2012/24797-2, 2014/10557-5FAPES

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