Droplet-based Microfluidic Systems Research Articles

Directed Evolution in Drops: Molecular Aspects and Applications.

The process of optimizing the properties of biological molecules is paramount for many industrial and medical applications. Directed evolution is a powerful technique for modifying and improving biomolecules such as proteins or nucleic acids (DNA or RNA). Mimicking the mechanism of natural evolution, one can enhance a desired property by applying a suitable selection pressure and sorting improved variants. Droplet-based microfluidic systems offer a high-throughput solution to this approach by helping to overcome the limiting screening steps and allowing the analysis of variants within increasingly complex libraries. Here, we review cases where successful evolution of biomolecules was achieved using droplet-based microfluidics, focusing on the molecular processes involved and the incorporation of microfluidics to the workflow. We highlight the advantages and limitations of these microfluidic systems compared to low-throughput methods and show how the integration of these systems into directed evolution workflows can open new avenues to discover or improve biomolecules according to user-defined conditions.

Recent advances of integrated microfluidic suspension cell culture system.

Microfluidic devices with superior microscale fluid manipulation ability and large integration flexibility offer great advantages of high throughput, parallelisation and multifunctional automation. Such features have been extensively utilised to facilitate cell culture processes such as cell capturing and culturing under controllable and monitored conditions for cell-based assays. Incorporating functional components and microfabricated configurations offered different levels of fluid control and cell manipulation strategies to meet diverse culture demands. This review will discuss the advances of single-phase flow and droplet-based integrated microfluidic suspension cell culture systems and their applications for accelerated bioprocess development, high-throughput cell selection, drug screening and scientific research to insight cell biology. Challenges and future prospects for this dynamically developing field are also highlighted.

Microfluidics-Integrated Sensors toward Rapid Detection of Single Nucleotide Variations.

Rapid detection of single nucleotide variations (SNVs) is of critical importance to early diagnosis of several diseases and the prediction of diverse responses to a specific treatment. Based on the information published in the literature, discrimination of SNVs is a developing area of study with great research enthusiasm and is also an area that can benefit from microfluidics-integrated designs. This review provides a brief overview of different microfluidics-based strategies for rapid detection of SNVs and mismatched bases. Sensors based on various microfluidic formats, such as paper-based microfluidic biosensors, droplet-based microfluidic systems, and magnetic bead-based microfluidic biosensors, have been discussed with respect to their specific pros and cons for SNV detection. These systems have shown promise for distributed on-site diagnostics in personalized medicine.

Controlled self-assembly of microgels in microdroplets

Microgels with embedded distinct compartments are robust tools for creating different physiochemical scaffolding conditions by co-encapsulation of various cargoes. This work presents a robust, rapid, and low-cost approach for one-step fabrication of multicompartmental microgels using droplet-based microfluidic systems. After selective modification of microchannels wettability using a simple and reliable patterning approach, double emulsion (DE) droplets are generated with desired number, size and composition of internal precursor core droplets. Upon gelation, the microgels confined in each oil droplet self-assemble, followed by their rapid release from oil droplet. By tuning the droplet generation and crosslinking parameters, complex microgels with desired structure and composition are fabricated to demonstrate the potential of the method for flexible production of codded microgels. The DE approach applied here enables accurate confinement of target compartments, provides a clog-free on-chip gelation, and more importantly offers high biocompatibility due to rapid release of microgels from unfavorable reagents. Implementation of different hydrogels such as alginate, agarose, and gelatin methacryloyl further demonstrates the robustness of presented method which is not limited to a specific material or gelling condition. This method can provide new opportunities in many biomedical applications from tissue engineering to drug delivery and high-throughput multiplex bioanalysis.

In Situ X-ray Absorption Spectroscopy and Droplet-Based Microfluidics: An Analysis of Calcium Carbonate Precipitation.

Droplet-based microfluidic systems are ideally suited for the investigation of nucleation and crystallization processes. To best leverage the features of such platforms (including exquisite time resolution and high-throughput operation), sensitive and in situ detection schemes are needed to extract real-time chemical information about all species of interest. In this regard, the extension of conventional (UV, visible, and infrared) optical detection schemes to the X-ray region of the electromagnetic spectrum is of high current interest, as techniques such as X-ray absorption spectroscopy (XAS) provide for the element-specific investigation of the local chemical environment. Accordingly, herein, we report for the first time the integration of millisecond droplet-based microfluidics with XAS. Such a platform allows for the sensitive acquisition of X-ray absorption data from picoliter-volume droplets moving at high linear velocities. Significantly, the high-temporal resolution of the droplet-based microfluidic platform enables unprecedented access to the early stages of the reaction. Using such an approach, we demonstrate in situ monitoring of calcium carbonate precipitation by extracting XAS spectra at the early time points of the reaction with a dead time as low as 10 ms. We obtain insights into the kinetics of the formation of amorphous calcium carbonate (ACC) as a first species during the crystallization process by monitoring the proportion of calcium ions converted into ACC. Within the confined and homogeneous environment of picoliter-volume droplets, the ACC content reaches 60% over the first 130 ms. More generally, the presented method offers new opportunities for the real-time monitoring of fast chemical and biological processes.

Spontaneous motion of droplet in tubes: The effects of surface wettability and segment length

This work studies the spontaneous droplet flow in tubes with different wettability gradient and number of segments by using a particle-based numerical method, many-body dissipative particle dynamics (MDPD). The study aims to understand how the different contact angle sets (gradient contact angle distribution along with different segments of a tube) and the number of segments could affect the droplet flow velocity and time to reaching a stable state. Simulation results show that even with the same wettability gap, a contact angle set with higher values of contact angle along the tube segments can drive the droplet to flow faster in the tubes; moreover, with the same contact angle set and tube length, the tubes with fewer segments can allow a faster flow of droplet, thus a shorter time to reaching the stable state. The numerical findings in this work can provide a new idea and direction for the design of droplet-based microfluidic systems.

Gas Crosstalk between PFPE-PEG-PFPE Triblock Copolymer Surfactant-Based Microdroplets and Monitoring Bacterial Gas Metabolism with Droplet-Based Microfluidics.

The PFPE–PEG–PFPE (Perfluoropolyether-polyethylene glycol-perfluoropolyether) surfactant has been used in droplet-based microfluidics and is known to provide high droplet stability and biocompatibility. Since this surfactant ensures the stability of droplets, droplet-based microfluidic systems have been widely used to encapsulate and analyze various biological components at the single-molecule scale, including viruses, bacteria, nucleic acids and proteins. In this study, we experimentally confirmed that gas crosstalk occurred between droplets formed by fluorinated oil and the PFPE–PEG–PFPE surfactant. E. coli K-12 bacterial cells were encapsulated with Luria–Bertani broth within droplets for the cultivation, and gas crosstalk was identified with neighboring droplets that contain phenol red. Since bacteria produce ammonia gas during its metabolism, penetration of ammonia gas initiates a color change of phenol red-containing droplets. Ammonia gas exchange was also confirmed by reacting ammonium chloride and sodium hydroxide within droplets that encapsulated. Herein, we demonstrate the gas crosstalk issue between droplets when it is formed using the PFPE–PEG–PFPE surfactant and also confirm that the density of droplet barrier has effects on gas crosstalk. Our results also suggest that droplet-based microfluidics can be used for the monitoring of living bacteria by the determination of bacterial metabolites during cultivation.

Theoretical and experimental limits of monodisperse droplet generation

Droplet microfluidic systems are becoming routine in advanced biochemical studies such as single cell gene expression, immuno profiling, precise nucleic acid quantification (dPCR) and particle synthesis. For all these applications, ensuring droplet monodispersity is critical to minimize the uncertainty due to droplet volume variation. Despite the wide usage of droplet-based microfluidic systems, the limit of monodispersity for droplet generation systems is still unknown. Here, we present an analytical approach that takes into account all the system dynamics and internal/external factors that disturb monodispersity. Interestingly, we are able to model the dynamics of a segmented two-phase flow system using a single-phase flow analogy, electron flow, in electrical circuits. We offer a unique solution and design guidelines to ensure ultra-monodisperse droplet generation. Our analytical conclusions are experimentally verified using a T-junction droplet generator. Equally importantly, we show the limiting experimental factors for reaching the theoretical maximum of monodispersity.

Breaking the Interface: Efficient Extraction of Magnetic Beads from Nanoliter Droplets for Automated Sequential Immunoassays.

Droplet-based microfluidic systems offer a high potential for miniaturization and automation. Therefore, they are becoming an increasingly important tool in analytical chemistry, biosciences, and medicine. Heterogeneous assays commonly utilize magnetic beads as a solid phase. However, the sensitivity of state of the art microfluidic systems is limited by the high bead concentrations required for efficient extraction across the water-oil interface. Furthermore, current systems suffer from a lack of technical solutions for sequential measurements of multiple samples, limiting their throughput and capacity for automation. Taking advantage of the different wetting properties of hydrophilic and hydrophobic areas in the channels, we improve the extraction efficiency of magnetic beads from aqueous nanoliter-sized droplets by 2 orders of magnitude to the low μg/mL range. Furthermore, the introduction of a switchable magnetic trap enables repetitive capture and release of magnetic particles for sequential analysis of multiple samples, enhancing the throughput. In comparison to conventional ELISA-based sandwich immunoassays on microtiter plates, our microfluidic setup offers a 25-50-fold reduction of sample and reagent consumption with up to 50 technical replicates per sample. The enhanced sensitivity and throughput of this system open avenues for the development of automated detection of biomolecules at the nanoliter scale.

Generation and manipulation of oil-in-water micro-droplets by confined thermocapillary microvortices.

Optofluidic manipulation of droplets is critical in droplet-based microfluidic systems for chemistry, biology, and medicine. Here, we reported a thermocapillary microvortices-based manipulation platform for controlling oil-in-water droplets through integrating a photothermal waveguide into a microfluidic chip. The sizes and shapes of the droplets can be controlled by adjusting optical power or positions of the water-oil interface. Here, teardrop-shaped droplets, which can encapsulate and accumulate mesoscopic matters easily, were generated when the water-oil interface and the channel boundaries approached the photothermal waveguide center simultaneously. The results showed that the thermocapillary microvortices have good controllability of droplet positions, droplet volumes, and encapsulated-particle distribution and thus it will be a powerful droplet manipulation strategy for microreactors and microcapsules.

Nanosensors-Assisted Quantitative Analysis of Biochemical Processes in Droplets.

Here, we present a miniaturized lab-on-a-chip detecting system for an all-electric and label-free analysis of the emulsion droplets incorporating the nanoscopic silicon nanowires-based field-effect transistors (FETs). We specifically focus on the analysis of β-galactosidase e.g., activity, which is an important enzyme of the glycolysis metabolic pathway. Furthermore, the efficiency of the synthesis and action of β-galactosidase can be one of the markers for several diseases, e.g., cancer, hyper/hypoglycemia, cell senescence, or other disruptions in cell functioning. We measure the reaction and reaction kinetics-associated shift of the source-to-drain current Isd in the system, which is caused by the change of the ionic strength of the microenvironment. With these results, we demonstrate that the ion-sensitive FETs are able to sense the interior of the aqueous reactors; thus, the conjunction of miniature nanosensors and droplet-based microfluidic systems conceptually opens a new route toward a sensitive, optics-less analysis of biochemical processes.

Mass spec sorts droplets

With droplet-based microfluidic systems, researchers can rapidly screen compound and reaction libraries. These systems often use fluorescent reporters to help separate compounds and reaction conditions that researchers want from those they don’t. The problem with relying on fluorescence is that most industrially relevant reactions don’t involve fluorescent reactants or products. Researchers led by Shuwen Sun and Jeffrey C. Moore of Merck & Co. and Robert T. Kennedy of the University of Michigan have now combined droplet-based screening systems with mass spectrometry–activated sorting to overcome that constraint (Angew. Chem., Int. Ed. 2019, DOI: 10.1002/anie.201913203). “Merck has had a profound interest in high-throughput screening in various forms for a long time,” Moore says. The company is especially interested in using such systems to screen for new enzyme catalysts as part of a directed evolution program. But few of the compounds in such a program are suitable for fluorescence-based sorting. The new screening

Droplet microfluidics: fundamentals and its advanced applications.

Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors, resulting in decreased reaction times. This, coupled with the precise generation and repeatability of droplet operations, has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as micro-reactors ranging from the nano- to femtoliter (10−15 liters) range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. For this, in the following article we will focus on the various droplet operations, as well as the numerous applications of the system and its future in many advanced scientific fields. Due to advantages of droplet-based systems, this technology has the potential to offer solutions to today's biomedical engineering challenges for advanced diagnostics and therapeutics.

Droplet-based optofluidic systems for measuring enzyme kinetics.

The study of enzyme kinetics is of high significance in understanding metabolic networks in living cells and using enzymes in industrial applications. To gain insight into the catalytic mechanisms of enzymes, it is necessary to screen an enormous number of reaction conditions, a process that is typically laborious, time-consuming, and costly when using conventional measurement techniques. In recent times, droplet-based microfluidic systems have proved themselves to be of great utility in large-scale biological experimentation, since they consume a minimal sample, operate at high analytical throughput, are characterized by efficient mass and heat transfer, and offer high levels of integration and automation. The primary goal of this review is the introduction of novel microfluidic tools and detection methods for use in high-throughput and sensitive analysis of enzyme kinetics. The first part of this review focuses on introducing basic concepts of enzyme kinetics and describing most common microfluidic approaches, with a particular focus on segmented flow. Herein, the key advantages include accurate control over the flow behavior, efficient mass and heat transfer, multiplexing, and high-level integration with detection modalities. The second part describes the current state-of-the-art platforms for high-throughput and sensitive analysis of enzyme kinetics. In addition to our categorization of recent advances in measuring enzyme kinetics, we have endeavored to critically assess the limitations of each of these detection approaches and propose strategies to improve measurements in droplet-based microfluidics. Graphical abstract.

CO2 Laser Fabrication of PMMA Microfluidic Double T-Junction Device with Modified Inlet-Angle for Cost-Effective PCR Application.

The formation of uniform droplets and the control of their size, shape and monodispersity are of utmost importance in droplet-based microfluidic systems. The size of the droplets is precisely tuned by the channel geometry, the surface interfacial tension, the shear force and fluid velocity. In addition, the fabrication technique and selection of materials are essential to reduce the fabrication cost and time. In this paper, for reducing the fabrication cost Polymethyl methacrylate (PMMA) sheet is used with direct write laser technique by VERSA CO2 laser VLS3.5. This laser writing technique gives minimum channel width of about , which limit miniaturizing the droplet. To overcome this, modification on double T-junction (DTJ) channel geometry has been done by modifying the channel inlets angles. First, a two-dimensional (2D) simulation has been done to study the effect of the new channel geometry modification on droplet size, droplets distribution inside the channel, and its throughput. The fabricated modified DTJ gives the minimum droplet diameter of , while DTJ channel produced droplet diameter of at the same conditions. Moreover, the modified double T-junction (MDTJ) decreases the variation in droplets diameter at the same flow rates by than DTJ. This low variation in the droplet diameter is suitable for repeatability of the DNA detection results. The MDTJ also enhanced the droplet generation frequency by more than the DTJ channel. The uniformity of droplet distribution inside the channel was enhanced by compared to the DTJ channel geometry. This fabrication technique eliminates the need for a photomask and cleanroom environment in addition shortening the cost and time. It takes only for fabrication. The minimum generated droplet diameter is within with more than droplets per second (at . oil flow rate). The device is a high-throughput and low-cost micro-droplet formation aimed to be as a front-end to a dynamic droplet digital PCR (ddPCR) platform for use in resource-limited environment.

Recent Advances in Droplet-based Microfluidic Technologies for Biochemistry and Molecular Biology.

Recently, droplet-based microfluidic systems have been widely used in various biochemical and molecular biological assays. Since this platform technique allows manipulation of large amounts of data and also provides absolute accuracy in comparison to conventional bioanalytical approaches, over the last decade a range of basic biochemical and molecular biological operations have been transferred to drop-based microfluidic formats. In this review, we introduce recent advances and examples of droplet-based microfluidic techniques that have been applied in biochemistry and molecular biology research including genomics, proteomics and cellomics. Their advantages and weaknesses in various applications are also comprehensively discussed here. The purpose of this review is to provide a new point of view and current status in droplet-based microfluidics to biochemists and molecular biologists. We hope that this review will accelerate communications between researchers who are working in droplet-based microfluidics, biochemistry and molecular biology.

Advances in the Use of Microfluidics to Study Crystallization Fundamentals.

This review compares droplet-based microfluidic systems used to study crystallization fundamentals in chemistry and biology. An original high-throughput droplet-based microfluidic platform is presented. It uses nanoliter droplets, generates a chemical library, and directly solubilizes powder, thus economizing both material and time. It is compatible with all solvents without the need for surfactant. Its flexibility permits phase diagram determination and crystallization studies (screening and optimizing experiments) and makes it easy to use for nonspecialists in microfluidics. Moreover, it allows concentration measurement via ultraviolet spectroscopy and solid characterization via X-ray diffraction analysis.

One-step wettability patterning of PDMS microchannels for generation of monodisperse alginate microbeads by in Situ external gelation in double emulsion microdroplets

Droplet-based microfluidic systems are promising tools for many biological applications from digitalized analysis to drug delivery and biomolecules synthesis. Among these, fabrication of microgels, particularly alginate microbeads, has substantial importance due to their capability for three-dimensional (3D) encapsulation of cells, drugs and other bioactive materials. However, biocompatible generation of monodisperse alginate microbeads is still challenging. In this study, a new droplet-based microfluidic strategy is developed for cell and chip friendly fabrication of monodispersed alginate microbeads. The polydimethylsiloxane (PDMS) microfluidic device is first treated using on-chip plasma-assisted deposition of polyvinyl alcohol, to selectively modify the hydrophobicity of microchannels. The proposed wettability patterning approach is permanent, simple, reliable, and time-effective, which makes the resulting microfluidic device highly stable and controllable for generation of double emulsions (DEs). The device is then used for fabrication of monodisperse alginate microbeads using external gelation in DE microdroplets. The reported microbead fabrication method offers both biocompatibility as a result of external gelation and rapid recovery of beads from processing solutions, as well as chip-compatibility due to clog-free gelation. This simple and robust DE strategy provides new opportunities not only for 3D micro-encapsulation but also for other droplet-based microfluidic applications in diagnostic testing and drug delivery.

A Taylor analogy model for droplet dynamics in planar extensional flow

The dynamics of a droplet suspended in a medium fluid is determined by the hydrodynamic forces and surface tension force, and the Taylor analogy had been successfully employed to predict droplet deformation and breakup in a spray. This paper aims to extend the Taylor analogy for prediction of droplet dynamics in planar extensional flow which has great significance in droplet-based microfluidic systems. Performance of the proposed model is compared with the three-dimensional numerical simulation results over a wide range of capillary number and viscosity ratio. Experimental data available in the literature is also compared with the prediction results for verification purpose. The proposed model could describe both the time scale and the magnitude of droplet deformation accurately.

Droplet‐based microfluidics systems in biomedical applications

Microfluidics has made a very impressive progress in the past decades due to its unique and instinctive advantages. Droplet-based microfluidic systems show excellent compatibility with many chemical and biological reagents and are capable of performing variety of operations that can implement microreactor, complex multiple core-shell structure, and many applications in biomedical research such as drug encapsulation, targeted drug delivery systems, and multifunctionalization on carriers. Droplet-based systems have been directly used to synthesize particles and encapsulate many biological entities for biomedicine applications due to their powerful encapsulation capability and facile versatility. In this paper, we review its origin, deviation, and evolution to draw a clear future, especially for droplet-based biomedical applications. This paper will focus on droplet generation, variations and complication as starter, and logistically lead to the numerous typical applications in biomedical research. Finally, we will summarize both its challenge and future prospects relevant to its droplet-based biomedical applications.