On‐Site Formation of Emulsions by Controlled Air Plugs

Air plugs are usually undesirable in microfluidic systems because of their detrimental effect on the system's stability and integrity. By controlling the wetting properties as well as the topographical geometry of the microchannel, it is reported herein that air plugs can be generated in pre‐defined locations to function as a unique valve, allowing for the on‐site formation of various emulsions including single‐component droplets, composite droplets with droplet‐to‐droplet concentration gradient, blood droplets, paired droplets, as well as bubble arrays without the need for precious flow control, a difficult task with conventional droplet microfluidics. Moreover, the self‐generated air valve can be readily deactivated (turned off) by the introduction of an oil phase, allowing for the on‐demand release of as‐formed droplets for downstream applications. It is proposed that the simple, yet versatile nature of this technique can act as an important method for droplet microfluidics and, in particular, is ideal for the development of affordable lab‐on‐a‐chip systems without suffering from scalability and manufacturing challenges that typically confound the conventional droplet microfluidics.     

A New Approach to In‐Situ “Micromanufacturing”: Microfluidic Fabrication of Magnetic and Fluorescent Chains Using Chitosan Microparticles as Building Blocks

An in situ microfluidic assembly approach is described that can both produce microsized building blocks and assemble them into complex multiparticle configurations in the same microfluidic device. The building blocks are microparticles of the biopolymer chitosan, which is intentionally selected because its chemistry allows for simultaneous intraparticle and interparticle linking. Monodisperse chitosan‐bearing droplets are created by shearing off a chitosan solution at a microfluidic T‐junction with a stream of hexadecane containing a nonionic detergent. These droplets are then interfacially crosslinked into stable microparticles by a downstream flow of glutaraldehyde (GA). The functional properties of these robust microparticles can be easily varied by introducing various payloads, such as magnetic nanoparticles and/or fluorescent dyes, into the chitosan solution. The on‐chip connection of such individual particles into well‐defined microchains is demonstrated using GA again as the chemical “glue” and microchannel confinement as the spatial template. Chain flexibility can be tuned by adjusting the crosslinking conditions: both rigid chains and semiflexible chains are created. Additionally, the arrangement of particles within a chain can also be controlled, for example, to generate chains with alternating fluorescent and nonfluorescent microparticles. Such microassembled chains could find applications as microfluidic mixers, delivery vehicles, microscale sensors, or miniature biomimetic robots.     

Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay

We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of β-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold.     

Thermoelectric manipulation of aqueous droplets in microfluidic devices

This article describes a method for manipulating the temperature inside aqueous droplets, utilizing a thermoelectric cooler to control the temperature of select portions of a microfluidic chip. To illustrate the adaptability of this approach, we have generated an "ice valve" to stop fluid flow in a microchannel. By taking advantage of the vastly different freezing points for aqueous solutions and immiscible oils, we froze a stream of aqueous droplets that were formed on-chip. By integrating this technique with cell encapsulation into aqueous droplets, we were also able to freeze single cells encased in flowing droplets. Using a live-dead stain, we confirmed the viability of cells was not adversely affected by the process of freezing in aqueous droplets provided cryoprotectants were utilized. When combined with current droplet methodologies, this technology has the potential to both selectively heat and cool portions of a chip for a variety of droplet-related applications, such as freezing, temperature cycling, sample archiving, and controlling reaction kinetics.     

Coding of Experimental Conditions in Microfluidic Droplet Assays Using Colored Beads and Machine Learning Supported Image Analysis

To efficiently exploit the potential of several millions of droplets that can be considered as individual bioreactors in microfluidic experiments, methods to encode different experimental conditions in droplets are needed. The approach presented here is based on coencapsulation of colored polystyrene beads with biological samples. The decoding of the droplets, as well as content quantification, are performed by automated analysis of triggered images of individual droplets in‐flow using bright‐field microscopy. The decoding strategy combines bead classification using a random forest classifier and Bayesian inference to identify different codes and thus experimental conditions. Antibiotic susceptibility testing of nine different antibiotics and the determination of the minimal inhibitory concentration of a specific antibiotic against a laboratory strain of Escherichia coli are presented as a proof‐of‐principle. It is demonstrated that this method allows successful encoding and decoding of 20 different experimental conditions within a large droplet population of more than 10⁵ droplets per condition. The decoding strategy correctly assigns 99.6% of droplets to the correct condition and a method for the determination of minimal inhibitory concentration using droplet microfluidics is established. The current encoding and decoding pipeline can readily be extended to more codes by adding more bead colors or color combinations.     

Mixing crowded biological solutions in milliseconds

In vitro studies of biological reactions are rarely performed in conditions that reflect their native intracellular environments where macromolecular crowding can drastically change reaction rates. Kinetics experiments require reactants to be mixed on a time scale faster than that of the reaction. Unfortunately, highly concentrated solutions of crowding agents such as bovine serum albumin and hemoglobin that are viscous and sticky are extremely difficult to mix rapidly. We demonstrate a new droplet-based microfluidic mixer that induces chaotic mixing of crowded solutions in milliseconds due to protrusions of the microchannel walls that generate oscillating interfacial shear within the droplets. Mixing in the microfluidic mixer is characterized, mechanisms underlying mixing are discussed, and evidence of biocompatibility is presented. This microfluidic platform will allow for the first kinetic studies of biological reactions with millisecond time resolution under conditions of macromolecular crowding similar to those within cells.     

Design of a Microchannel‐Nanochannel‐Microchannel Array Based Nanoelectroporation System for Precise Gene Transfection

A micro/nano‐fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non‐viral gene transfection is described. A dip‐combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4‐butanediol diacrylate (1,4‐BDDA), adopted to convert the microridge‐nanowire‐microridge array into a microchannel‐nanochannel‐microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 μm to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow‐up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.     

Formation of droplets of alternating composition in microfluidic channels and applications to indexing of concentrations in droplet-based assays

For screening the conditions for a reaction by using droplets (or plugs) as microreactors, the composition of the droplets must be indexed. Indexing here refers to measuring the concentration of a solute by addition of a marker, either internal or external. Indexing may be performed by forming droplet pairs, where in each pair the first droplet is used to conduct the reaction, and the second droplet is used to index the composition of the first droplet. This paper characterizes a method for creating droplet pairs by generating alternating droplets, of two sets of aqueous solutions in a flow of immiscible carrier fluid within PDMS and glass microfluidic channels. The paper also demonstrates that the technique can be used to index the composition of the droplets, and this application is illustrated by screening conditions of protein crystallization. The fluid properties required to form the steady flow of the alternating droplets in a microchannel were characterized as a function of the capillary number Ca and water fraction. Four regimes were observed. At the lowest values of Ca, the droplets of the two streams coalesced; at intermediate values of Ca the alternating droplets formed reliably. At even higher values of Ca, shear forces dominated and caused formation of droplets that were smaller than the cross-sectional dimension of the channel; at the highest values of Ca, coflowing laminar streams of the two immiscible fluids formed. In addition to screening of protein crystallization conditions, understanding of the fluid flow in this system may extend this indexing approach to other chemical and biological assays performed on a microfluidic chip.     

Breath Figures as a Dynamic Templating Method for Polymers and Nanomaterials

This review describes the use of breath figures as a templating method for the fabrication of self‐assembled polymeric‐ and nanoparticle‐based micro‐ and nanostructures. If moist air is blown over a solution of a polymer or stabilized nanoparticles in an organic solvent, such as carbon disulfide, benzene, or chloroform, evaporative cooling leads to the formation of water droplets on the liquid surface. The monodisperse droplets arrange into a hexagonal array and sink into the polymer solution. Removal of the solvent and the water leaves an imprint of the water droplets as a hollow, air‐filled, hexagonally ordered, polymeric bubble array. Progress in the field of breath‐figure formation is reviewed. The application of breath figures for the generation of functional structures in chemistry and materials science is discussed.     

Sequential Coalescence Enabled Two‐Step Microreactions in Triple‐Core Double‐Emulsion Droplets Triggered by an Electric Field

Advances in microfluidic emulsification have enabled the generation of exquisite multiple‐core droplets, which are promising structures to accommodate microreactions. An essential requirement for conducting reactions is the sequential coalescence of the multiple cores encapsulated within these droplets, therefore, mixing the reagents together in a controlled sequence. Here, a microfluidic approach is reported for the conduction of two‐step microreactions by electrically fusing three cores inside double‐emulsion droplets. Using a microcapillary glass device, monodisperse water‐in‐oil‐in‐water droplets are fabricated with three compartmented reagents encapsulated inside. An AC electric field is then applied through a polydimethylsiloxane chip to trigger the sequential mixing of the reagents, where the precise sequence is guaranteed by the discrepancy of the volume or conductivity of the inner cores. A two‐step reaction in each droplet is ensured by two times of core coalescence, which totally takes 20–40 s depending on varying conditions. The optimal parameters of the AC signal for the sequential fusion of the inner droplets are identified. Moreover, the capability of this technique is demonstrated by conducting an enzyme‐catalyzed reaction used for glucose detection with the double‐emulsion droplets. This technique should benefit a wide range of applications that require multistep reactions in micrometer scale.     

A Counter Propagating Lens‐Mirror System for Ultrahigh Throughput Single Droplet Detection

Fluorescence‐based detection schemes provide for multiparameter analysis in a broad range of applications in the chemical and biological sciences. Toward the realization of fully portable analysis systems, microfluidic devices integrating diverse functional components have been implemented in a range of out‐of‐lab environments. That said, there still exits an unmet and recognized need for miniaturized, low‐cost, and sensitive optical detection systems, which provide not only for efficient molecular excitation, but also enhanced photon collection capabilities. To this end, an optofluidic platform that is adept at enhancing fluorescence light collection from microfluidic channels is presented. The central component of the detection module is a monolithic parabolic mirror located directly above the microfluidic channel, which acts to enhance the number of emitted photons reflected toward the detector. In addition, two‐photon polymerization is used to print a microscale‐lens below the microfluidic flow channel and directly opposite the mirror, to enhance the delivery of excitation radiation into the channel. Using such an approach, it is demonstrated that fluorescence signals can be enhanced by over two orders of magnitude, with component parallelization enabling the detection of pL‐volume droplets at rates up to 40 000 droplets per second.     

Scalable Production of Biomedical Microparticles via High-Throughput Microfluidic Step Emulsification

Drug microcarriers are widely used in disease treatment, and microfluidics is well established in the preparation of microcarrier particles. A proper design of the microfluidic platform toward scalable production of drug microcarriers can extend its application values in wound healing, where large numbers of microcarriers are required. Here, a microfluidic step emulsification method for the preparation of monodisperse droplets is presented. The droplet size depends primarily on the microchannel depth rather than flow rate, making the system robust for high-throughput production of droplets and hydrogel microparticles. Based on this platform, basic fibroblast growth factor (bFGF) is uniformly encapsulated in the microparticles, and black phosphorus (BP) is incorporated for controllable release via near-infrared (NIR) stimulation. The microparticles serve as drug carriers to be applied to the wound site, inducing angiogenesis and collagen deposition, thereby accelerating wound repair. These results indicate that the step emulsification technique provides a promising solution to scalable production of drug microcarriers for wound healing as well as tissue regeneration.     

One Cell,One Drop,One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co‐planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL‐sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in‐house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI‐H1299 lung squamous cell population resulting from loss‐of‐function eGFP and RAF1 gene knockout transfections.     

Injectable Cardiac Cell Microdroplets for Tissue Regeneration

One of the strategies for heart regeneration includes cell delivery to the defected heart. However, most of the injected cells do not form quick cell–cell or cell–matrix interactions, therefore, their ability to engraft at the desired site and improve heart function is poor. Here, the use of a microfluidic system is reported for generating personalized hydrogel‐based cellular microdroplets for cardiac cell delivery. To evaluate the system's limitations, a mathematical model of oxygen diffusion and consumption within the droplet is developed. Following, the microfluidic system's parameters are optimized and cardiac cells from neonatal rats or induced pluripotent stem cells are encapsulated. The morphology and cardiac specific markers are assessed and cell function within the droplets is analyzed. Finally, the cellular droplets are injected to mouse gastrocnemius muscle to validate cell retention, survival, and maturation within the host tissue. These results demonstrate the potential of this approach to generate personalized cellular microtissues, which can be injected to distinct regions in the body for treating damaged tissues.     

Design and analysis of single-cell capture microfluidic device

The aim of our study was to develop microfluidic devices using microchannel technology with the capability of capturing single cells. We analyzed and compared the cell-capturing efficiencies of series-loop microchannel and parallel-loop microchannel devices that were produced using polydimethylsiloxane (PDMS). Each set of microchannels was composed of a main flow channel and several branch channels with capturing zones. The microfluidic devices were designed to use the differences in flow rates between the main flow channel and the branch channels as a means of capturing single cells based on size and sequestering them within the microstructure of multiple capture zones. The data indicated that the flow medium encountered significant resistance in the series-loop microchannel device, which resulted in an inability to hold the captured cells within any of the capture zones. Flow resistance was, however, greatly reduced in the parallel-loop microchannel device compared to the series-loop device, and single cells were captured in all the capturing zones of the device. Our data suggest that the parallel-loop microchannel technology has significant potential for development toward high-throughput platforms capable of capturing single cells for physiological analyses at the single-cell level.     

On-demand patterning of protein matrixes inside a microfluidic device

On-demand immobilization of proteins at specific locations in a microfluidic device would advance many types of bioassays. We describe a strategy to create a patterned surface within a microfluidic channel by electrochemical means, which enables site-specific immobilization of protein matrixes and cells under physiological conditions, even after the device is fully assembled. By locally generating hypobromous acid at a microelectrode in the microchannel, the heparin-coated channel surface rapidly switches from antibiofouling to protein-adhering. Since this transformation allows compartmentalizing of multiple types of antibodies into distinct regions throughout the single microchannel, simultaneous assay of two kinds of complementary proteins was possible. This patterning procedure can be applied to conventional microfluidic devices since it requires only some electrodes and a voltage source (1.7 V, DC).     

Liquid Metal–Based Soft Microfluidics

Motivated by the increasing demand of wearable and soft electronics, liquid metal (LM)‐based microfluidics has been subjected to tremendous development in the past decade, especially in electronics, robotics, and related fields, due to the unique advantages of LMs that combines the conductivity and deformability all‐in‐one. LMs can be integrated as the core component into microfluidic systems in the form of either droplets/marbles or composites embedded by polymer materials with isotropic and anisotropic distribution. The LM microfluidic systems are found to have broad applications in deformable antennas, soft diodes, biomedical sensing chips, transient circuits, mechanically adaptive materials, etc. Herein, the recent progress in the development of LM‐based microfluidics and their potential applications are summarized. The current challenges toward industrial applications and future research orientation of this field are also summarized and discussed.     

Generation of Femtoliter Reactor Arrays within a Microfluidic Channel for Biochemical Analysis

We present a simple microfluidic method to generate high-density femotoliter-sized microreactor arrays within microfluidic channels. In general, we designed a main channel with many small chambers built into its walls. After sequentially infusing aqueous solution and organic solvent from a single tube into the device, aqueous droplets are confined in the chambers by the solvent flow. The generated reactors are small and stable enough for carrying out ultrasensitive biochemical assays at single molecule levels. As a demonstration, in this paper, we optically observed hydrolysis activity of β-galactosidase enzymatic molecules in the reactor arrays at single molecule levels. Further, this method has the following advantages: (1) the droplets are observable immediately after formation and (2) its simple procedure is sufficiently robust such that even handy infusion of the preloaded solutions is reproducible. We believe our method provides a platform attractive to a variety of single molecule studies and sensing applications such as clinical diagnostics.     

Dynamics of capturing process of multiple magnetic nanoparticles in a flow through microfluidic bioseparation system

A mathematical model based on finite-element technique is developed for predicting the transport and capture of multiple magnetic nanoparticles in a microfluidic system that consists of a microfluidic channel enclosed by a permanent magnet. The trajectories and trapping efficiencies are calculated for multiple magnetic nanoparticles when released in the microsystem. It is demonstrated that not only the size but also the point of release of nanoparticles within the microchannel affects the capturing process. Influence of three important parameters, inlet velocities of fluid containing magnetic nanoparticles, diameter of magnetic nanoparticles and magnetic field strength on the trapping efficiency are investigated and optimised values of inlet velocity and magnetic field strength for completely trapping 50 nm magnetic nanoparticles are predicted. It is further demonstrated that the angular position of magnet around the microchannel is also critical in dictating the resulting bioseparation performance. Furthermore, combination of these analyses using the mathematical model will be very useful in the design and development of novel microfluidic bioseparation microsystems.     

Ultrathin Double‐Shell Capsules for High Performance Photon Upconversion

Triplet‐fusion‐based photon upconversion capsules with ultrathin double shells are developed through a single dripping instability in a microfluidic flow‐focusing device. An inner separation layer allows use of a brominated hydrocarbon oil‐based fluidic core, demonstrating significantly enhanced upconversion quantum yield. Furthermore, a perfluorinated photocurable monomer serves as a transparent shell phase with remote motion control through magnetic nanoparticle incorporation.