Droplet-based Microfluidic Technology Research Articles

Advances in Droplet-Based Microfluidic High-Throughput Screening of Engineered Strains and Enzymes Based on Ultraviolet, Visible, and Fluorescent Spectroscopy

Genetic engineering and directed evolution are effective methods for addressing the low yield and poor industrialization level of microbial target products. The current research focus is on how to efficiently and rapidly screen beneficial mutants from constructed large-scale mutation libraries. Traditional screening methods such as plate screening and well-plate screening are severely limited in their development and application due to their low efficiency and high costs. In the past decade, microfluidic technology has become an important high-throughput screening technology due to its fast speed, low cost, high automation, and high screening throughput, and it has developed rapidly. Droplet-based microfluidic high-throughput screening has been widely used in various fields, such as strain/enzyme activity screening, pathogen detection, single-cell analysis, drug discovery, and chemical synthesis, and has been widely applied in industries such as those involving materials, food, chemicals, textiles, and biomedicine. In particular, in the field of enzyme research, droplet-based microfluidic high-throughput screening has shown excellent performance in discovering enzymes with new functions as well as improved catalytic efficiency or stability, acid-base tolerance, etc. Currently, droplet-based microfluidic high-throughput screening technology has achieved the high-throughput screening of enzymes such as glycosidase, lipase, peroxidase, protease, amylase, oxidase, and transaminase as well as the high-throughput detection of products such as riboflavin, coumarin, 3-dehydroquinate, lactic acid, and ethanol. This article reviews the application of droplet-based microfluidics in high-throughput screening, with a focus on high-throughput screening strategies based on UV, visible, and fluorescence spectroscopy, including labeled optical signal detection screening, as well as label-free electrochemical detection, mass spectrometry, Raman spectroscopy, nuclear magnetic resonance, etc. Furthermore, the research progress and development trends of droplet-based microfluidic technology in enzyme modification and strain screening are also introduced.

Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes

The genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. However, existing single-cell methylomic technologies are based on use of tubes or well plates and these platforms are not easily scalable for handling a large number of single cells. Here we demonstrate a droplet-based microfluidic technology, Drop-BS, to construct single-cell bisulfite sequencing libraries for DNA methylome profiling. Drop-BS takes advantage of the ultrahigh throughput offered by droplet microfluidics to prepare bisulfite sequencing libraries of up to 10,000 single cells within 2 days. We apply the technology to profile mixed cell lines, mouse and human brain tissues to reveal cell type heterogeneity. Drop-BS offers a promising solution for single-cell methylomic studies requiring examination of a large cell population.

Synthesis and Characterization of Hydrogel Droplets Containing Magnetic Nano Particles, in a Microfluidic Flow-Focusing Chip.

Magnetic hybrid hydrogels have exhibited remarkable efficacy in various areas, particularly in the biomedical sciences, where these inventive substances exhibit intriguing prospects for controlled drug delivery, tissue engineering, magnetic separation, MRI contrast agents, hyperthermia, and thermal ablation. Additionally, droplet-based microfluidic technology enables the fabrication of microgels possessing monodisperse characteristics and controlled morphological shapes. Here, alginate microgels containing citrated magnetic nanoparticles (MNPs) were produced by a microfluidic flow-focusing system. Superparamagnetic magnetite nanoparticles with an average size of 29.1 ± 2.5 nm and saturation magnetization of 66.92 emu/g were synthesized via the co-precipitation method. The hydrodynamic size of MNPs was changed from 142 nm to 826.7 nm after the citrate group's attachment led to an increase in dispersion and the stability of the aqueous phase. A microfluidic flow-focusing chip was designed, and the mold was 3D printed by stereo lithographic technology. Depending on inlet fluid rates, monodisperse and polydisperse microgels in the range of 20-120 μm were produced. Different conditions of droplet generation in the microfluidic device (break-up) were discussed considering the model of rate-of-flow-controlled-breakup (squeezing). Practically, this study indicates guidelines for generating droplets with a predetermined size and polydispersity from liquids with well-defined macroscopic properties, utilizing a microfluidic flow-focusing device (MFFD). Fourier transform infrared spectrometer (FT-IR) results indicated a chemical attachment of citrate groups on MNPs and the existence of MNPs in the hydrogels. Magnetic hydrogel proliferation assay after 72 h showed a better rate of cell growth in comparison to the control group (p = 0.042).

Hundred-micron droplet ejection by focused ultrasound for genomic applications

Droplet-based microfluidic (DMF) technology is widely used in genetic analysis. Among multiple droplet generation techniques, acoustic droplet ejection (ADE) is a non-contact, high-precision method that utilizes the energy of focused ultrasound. The ejection of a single droplet of controlled size, the performance of multi-droplet ejections and the effects of the ultrasound energy on nucleic acid molecules in the droplets, are the main research interests in droplet-based genomics. In this work, a focused ultrasonic transducer with a frequency of 20 MHz was used, and we achieved droplet ejection successfully. Various experiments were carried out to study ADE and the beneficial results were obtained, including the characterization of ADE conditions, the relationship between droplet volume and parameters (including power, duration and distance), the energy required for ADE, and the effects of ultrasound energy on DNA strands, etc. It was first noticed that the superior limit of the droplet size could be increased by adjusting the height of the transducer. In addition, multi-droplet ejections at the optimal delay time were realized with good linearity. Above all, we demonstrated that the nucleic acid molecules remained their integrity when the exciting power was the maximum and the exciting duration was the longest in our system. In summary, ADE was proved to have an important role and a vast prospect in DMF technology for genomics-related applications.

Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening.

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based microfluidics technology, single cells can be encapsulated in droplets. The screening can be carried out by detecting the fluorescence intensity of secreted riboflavin. Thus, an efficient and high-throughput screening method suitable for riboflavin production strain improvement could be established. In this study, droplet-based microfluidics screening was applied, and a more competitive riboflavin producer U3 was selected from the random mutation library of strain S1. The riboflavin production and biomass of U3 were higher than that of S1 in flask fermentation. In addition, the results of fed-batch fermentation showed that the riboflavin production of U3 was 24.3 g/L, an 18% increase compared with the parent strain S1 (20.6 g/L), and the yield (g riboflavin/100 g glucose) increased by 19%, from 7.3 (S1) to 8.7 (U3). Two mutations of U3 (sinRG89R and icdD28E) were identified through whole genome sequencing and comparison. Then they were introduced into BS168DR (parent of S1) for further analysis, which also caused riboflavin production to increase. This paper provides protocols for screening riboflavin-producing B. subtilis with droplet-based microfluidics technology and reveals mutations in riboflavin overproduction strains.

Open-source tool for real-time and automated analysis of droplet-based microfluidic.

Droplet-based microfluidic technology is a powerful tool for generating large numbers of monodispersed nanoliter-sized droplets for ultra-high throughput screening of molecules or single cells. Yet further progress in the development of methods for the real-time detection and measurement of passing droplets is needed for achieving fully automated systems and ultimately scalability. Existing droplet monitoring technologies are either difficult to implement by non-experts or require complex experimentation setups. Moreover, commercially available monitoring equipment is expensive and therefore limited to a few laboratories worldwide. In this work, we validated for the first time an easy-to-use, open-source Bonsai visual programming language to accurately measure in real-time droplets generated in a microfluidic device. With this method, droplets are found and characterized from bright-field images with high processing speed. We used off-the-shelf components to achieve an optical system that allows sensitive image-based, label-free, and cost-effective monitoring. As a test of its use we present the results, in terms of droplet radius, circulation speed and production frequency, of our method and compared its performance with that of the widely-used ImageJ software. Moreover, we show that similar results are obtained regardless of the degree of expertise. Finally, our goal is to provide a robust, simple to integrate, and user-friendly tool for monitoring droplets, capable of helping researchers to get started in the laboratory immediately, even without programming experience, enabling analysis and reporting of droplet data in real-time and closed-loop experiments.

A novel high-throughput approach for transforming filamentous fungi employing a droplet-based microfluidic platform

Droplet-based microfluidic technology is a powerful tool for single-cell cultivation and rapid isolation of bacteria, yeasts and algae. However, it has been of limited use for studies of filamentous fungi due to the fast growth of their branched hyphae. The long regeneration time for fungal protoplasts and low-throughput screening methods are inherent problems for current genetic transformation techniques. Therefore, we have developed a novel droplet-based method for the filamentous fungus Trichoderma reesei expressing green fluorescent protein (GFP) as a marker. This approach presented several outstanding advantages over the traditional transformation method, including a 7-fold reduction in time for T. reesei protoplast regeneration, an 8-fold increase in regeneration frequency, and a screening speed of up to 8,000 droplets min−1. In this study, we encapsulated and incubated the gfp-transformed T. reesei protoplasts in droplets for 24 h, screened the droplets in a high-throughput assay, and eventually collected a transformant library with over 96 % of the candidates transformed with the marker gene. This versatile approach should make fungi more amenable to genetic manipulation and encourage strain improvements for industrial applications.

Collective behavior of crowded drops in microfluidic systems

In a crowded and confined system where many drops interact, how do the interactions give rise to collective behavior? How do these interactions impact the performance of droplet-based microfluidic technology, and how can we overcome or leverage such interactions? This paper reviews our recent work on answering these questions, by studying the collective behavior of droplets in a concentrated emulsion and tracking the dynamics and the fate of individual droplets within the emulsion in a microfluidic system.

A Novel Step-T-Junction Microchannel for the Cell Encapsulation in Monodisperse Alginate-Gelatin Microspheres of Varying Mechanical Properties at High Throughput.

Cell encapsulation has been widely employed in cell therapy, characterization, and analysis, as well as many other biomedical applications. While droplet-based microfluidic technology is advantageous in cell microencapsulation because of its modularity, controllability, mild conditions, and easy operation when compared to other state-of-art methods, it faces the dilemma between high throughput and monodispersity of generated cell-laden microdroplets. In addition, the lack of a biocompatible method of de-emulsification transferring cell-laden hydrogel from cytotoxic oil phase into cell culture medium also hurtles the practical application of microfluidic technology. Here, a novel step-T-junction microchannel was employed to encapsulate cells into monodisperse microspheres at the high-throughput jetting regime. An alginate–gelatin co-polymer system was employed to enable the microfluidic-based fabrication of cell-laden microgels with mild cross-linking conditions and great biocompatibility, notably for the process of de-emulsification. The mechanical properties of alginate-gelatin hydrogel, e.g., stiffness, stress–relaxation, and viscoelasticity, are fully adjustable in offering a 3D biomechanical microenvironment that is optimal for the specific encapsulated cell type. Finally, the encapsulation of HepG2 cells into monodisperse alginate–gelatin microgels with the novel microfluidic system and the subsequent cultivation proved the maintenance of the long-term viability, proliferation, and functionalities of encapsulated cells, indicating the promising potential of the as-designed system in tissue engineering and regenerative medicine.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC).

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remainat the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Field-induced formation and manipulation of a microscale liquid ball on an outside wall of the pulled pipette

We report a phenomenological observation of electric-field-induced formation and manipulation of liquid ball on an outer wall of the pulled pipette by using the quartz tuning fork-based atomic force microscope (QTF-AFM). The dye molecule solution with excitation wavelength of 488 nm and detection efficiency of 95% is used to investigate the movement characteristics of liquid droplets when the electric field is applied. The ejected liquid solution forms a microscale liquid droplet at the apex of the pipette by the application of electric field, containing dye molecules, which climbs up along the negatively charged outer surface of the pipette due to the electro-osmosis effect. With positive or negative bias voltages, we manipulate a liquid ball to slide upward or downward, respectively. This field-induced transport of a liquid droplet may be useful to nano-biotechnology or droplet-based microfluidic technology, for example, noncontact delivery and manipulation of liquid solution in the form of separated droplets.

Droplet microfluidic devices for organized stem cell differentiation into germ cells: capabilities and challenges.

The online version contains supplementary material available at 10.1007/s12551-021-00907-5.

Real-Time Single-Cell Monitoring of Drug Effects Using Droplet-Based Microfluidic Technology: A Proof-of-Concept Study.

Drugs that act on ribosome biogenesis and cell proliferation play important roles in treatment of human diseases. Moreover, measurement of drug effects at a single-cell level would create vast opportunities for pharmaceutical innovation. We present in this study an original proof-of-concept study of single-cell measurement of drug effects with a focus on inhibition of ribosome biogenesis and cell proliferation, and using yeast (Saccharomyces cerevisiae) as a model eukaryotic organism. We employed a droplet-based microfluidic technology and nucleolar protein-tagged strain of the yeast for real-time monitoring of the cells. We report a comprehensive account of the ways in which interrelated pathways are impacted by drug treatment in a single-cell level. Self-organizing maps, transcription factor, and Gene Ontology enrichment analyses were utilized to these ends. This article makes a contribution to advance single-cell measurement of drug effects. We anticipate the microfluidic technology platform presented herein is well poised for future applications in personalized/precision medicine research as well as in industrial settings for drug discovery and clinical development. In addition, the study offers new insights on ribosome biogenesis and cell proliferation that should prove useful in cancer research and other complex human diseases impacted by these key cellular processes.

Facile fabrication of drug-loaded PEGDA microcapsules for drug evaluation using droplet-based microchip

Droplet-based microfluidic technology can be utilized as a microreactor to prepare novel functional monodisperse microcapsules. In this study, a droplet-based microfluidic chip with surface modification, which allowed the one-step preparation of double emulsion microcapsules. An O/W/O double emulsion using polyethylene (glycol) diacrylate (PEGDA) solution as the intermediate water phase was prepared by regulating the hydrophilicity and hydrophobicity of the chip surface, with PEGDA microcapsules prepared using UV polymerization. And then anti-tumor drug paclitaxel and neurotoxin 6-OHDA were encapsulated in microcapsules for drug and toxicology evaluation, respectively. Compared to controls, drug-loaded microcapsules caused a significant increase in the death rate of PC12 cells. This indicates that the obtained drug-loaded microcapsules could be used in drug evaluation and potentially in drug screening and delivery.

Microfluidic production of monodisperse emulsions for cosmetics.

Droplet-based microfluidic technology has enabled the production of emulsions with high monodispersity in sizes ranging from a few to hundreds of micrometers. Taking advantage of this technology, attempts to generate monodisperse emulsion drops with high drug loading capacity, ordered interfacial structure, and multi-functionality have been made in the cosmetics industry. In this article, we introduce the practicality of the droplet-based microfluidic approach to the cosmetic industry in terms of innovation in productivity and marketability. Furthermore, we summarize some recent advances in the production of emulsion drops with enhanced mechanical interfacial stability. Finally, we discuss the future prospects of microfluidic technology in accordance with consumers' needs and industrial attributes.

Reading the writing of immunizations in mice – the quantitative assessment of secreted antibodies to evaluate the quality of immunizations

Abstract Vaccination needs to prepare the immune system to rapidly secrete specific antibodies upon a future encounter with the pathogen. Assessments of this feature are often challenging to perform, as measurements using antibody titers represent only a poorly resolved average of the rapidly evolving repertoire. We recently published and described a novel, droplet-based microfluidic technology that allows the quantitative characterization of humoral immune responses with single-cell and -antibody resolution. The employed snapshot analysis in this system enables studying even rapidly evolving repertoires. Additionally, the method extracts for each secreted antibody its production rate, specificity, and affinity, offering a high-resolution view of immunization. Consequently, this system enables the investigation of the immunization-generated secreted antibody repertoire with high temporal, analytical, and spatial resolution. This study in mice used the above method to characterize the quality of the immunization-induced immunoglobulin-G repertoire, and we correlated these measures with the recall-accessible repertoire. Here, we showed that introducing variation in the immunization schemes led to significant differences in the secreted repertoire. Additional analysis after recall allowed quantifying the accessible antibody repertoire, and due to the single-cell resolution, our data allowed us to compare these two repertoires on a phenotypical level. We showed that the relationships between the two repertoires are not trivial and strongly depend on immunization itself. In summary, the developed approach introduced a novel, quantitative, and functionally resolved alternative to study the quality of immunizations.

Droplet-Based Cytotoxicity Assay: Implementation of Time-Efficient Screening of Antitumor Activity of Natural Killer Cells

Natural killer (NK) cells are key players of the innate immune system. Due to their rapid cytotoxicity against infectious pathogens, hematologic malignancies, and solid tumors, NK cells represent solid candidates for cell-based immunotherapy. Despite the progress made in recent years, the heterogeneity in their cytotoxic behavior represents a drawback. With the goal of screening the intrinsic diversity of NK cells, droplet-based microfluidic technology is exploited to develop a single-cell time-efficient cytotoxicity assay. Toward this end, NK-92 cells are coencapsulated with hematological tumor cell lines in water-in-oil droplets of different sizes and their cytotoxic activity is evaluated. The effect of droplet-based confinement on NK cytotoxicity is investigated by controlling the droplet volume. The successful optimization of the droplet size allows for time efficiency compared to cytotoxicity assays based on flow cytometry. Additionally, the ability of individual NK-92 cells to kill multiple target cells in series is explored, expanding the knowledge about the serial killing process dynamics. The developed droplet-based microfluidic assay does not require the labeling of NK cells and represents a step toward developing of a forthcoming process for the selection of NK cells with the highest cytotoxicity against specific targets.

Biotherapeutics: Bone Scaffolds Based on Degradable Vaterite/PEG‐Composite Microgels (Adv. Healthcare Mater. 11/2020)

In article number 1901820 by Sebastian Seiffert and co-workers, degradable vaterite/PEG-composite microgels are templated by droplet-based microfluidic technology. A key aspect of the material development is the fundamental understanding of the gel structure-property relationships, and their connection to tissue engineering, by tailoring the microgel properties on micro-, meso-, and macroscopic levels. The co-encapsulation of cells provides an in vitro platform for metabolic bone regeneration studies and a step towards new bone tissue regeneration materials.

Eccentric magnetic microcapsule for on-demand transportation, release, and evacuation in microfabrication fluidic networks

In this study, an eccentric magnetic microcapsule is reported for on-demand transportation, release, and evacuation in a micro-fabrication fluidic network. Using a droplet-based microfluidic technology, monodisperse magnetic polydimethylsiloxane (PDMS) core-shell microcapsules with a small eccentric ferrofluid core and a big aqueous core are fabricated from templates of dual-cored water-in-oil-in-water ((W1+W2)/O/W) double-emulsion droplets. The eccentric ferrofluid core enables the microcapsule to have flexible magnetic responsibility and orientation property, and the aqueous core can realize the encapsulation and release of nanoliter-scale encapsulants. Each microcapsule can be magnetic-guided by an external magnetic field to travel in microchannels along a designated route, and thus transport the encapsulated cargo (the aqueous core) to the targeted region. Then, the promptly shell rupture of the microcapsule due to the action of a time-averaged electrical stress from Maxwell-Wagner structural polarization in an externally-imposed alternating-current (AC) electric field allows the release of cargo into the region. Moreover, the empty microcapsule after release of the aqueous core can be magnetic-guided to evacuate from the device. Finally, to prove the practical utility of the eccentric magnetic microcapsule for on-demand transportation and release of nanoliter-scale encapsulant, this microcapsule is used to targeted convey fluorescent nanoparticles, which not only can serve as tracers to show the release of the aqueous core, but also can be cargoes themselves for bioimaging, sensing, and so on.

Enhancing droplet transition capabilities using sloped microfluidic channel geometry for stable droplet operation.

Droplet-based microfluidics technology allows for the generation and control of droplets that function as independent chemical and biological reactors, enabling broad ranges of high-throughput assays. As more complex multi-step assays are being realized in droplet format, maintaining droplet stability throughout the assay becomes a critical requirement. Unfortunately, as droplets go through multiple manipulation steps, droplet breakage is commonly seen, especially where droplets have to go through sharp transitions in direction and shape. Standard microfabrication techniques typically result in inherent sharp geometry in Z-direction due to their two-dimensional fabrication nature. Recent advancement in micro- and nano- fabrication technology using two-photon polymerization (2PP) is enabling complex 3D microstructures with sub-micrometer resolution to be readily fabricated. Here, utilizing this microfabrication technique, we present a simple solution to the droplet stability challenge by utilizing sloped-geometry microfluidic channels to enable microdroplets to smoothly transition between microfluidic channels having two different heights without breakage. The technique and innovation demonstrated here have the potential to replace conventional droplet microfluidic device fabrication approaches and enable droplet microfluidic platforms to achieve significantly higher level of efficiency, accuracy, and stability never realized before.