Development and future of droplet microfluidics.

Over the past two decades, advances in droplet-based microfluidics have facilitated new approaches to process and analyze samples with unprecedented levels of precision and throughput. A wide variety of applications has been inspired across multiple disciplines ranging from materials science to biology. Understanding the dynamics of droplets enables optimization of microfluidic operations and design of new techniques tailored to emerging demands. In this review, we discuss the underlying physics behind high-throughput generation and manipulation of droplets. We also summarize the applications in droplet-derived materials and droplet-based lab-on-a-chip biotechnology. In addition, we offer perspectives on future directions to realize wider use of droplet microfluidics in industrial production and biomedical analyses.

High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2.

Mapping mutations and discovering cellular determinants that cause the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce infected cells to form syncytia would facilitate the development of strategies for blocking the formation of such cell-cell fusion. Here we describe high-throughput screening methods based on droplet microfluidics and the size-exclusion selection of syncytia, coupled with large-scale mutagenesis and genome-wide knockout screening via clustered regularly interspaced short palindromic repeats (CRISPR), for the large-scale identification of determinants of cell-cell fusion. We used the methods to perform deep mutational scans in spike-presenting cells to pinpoint mutable syncytium-enhancing substitutions in two regions of the spike protein (the fusion peptide proximal region and the furin-cleavage site). We also used a genome-wide CRISPR screen in cells expressing the receptor angiotensin-converting enzyme 2 to identify inhibitors of clathrin-mediated endocytosis that impede syncytium formation, which we validated in hamsters infected with SARS-CoV-2. Finding genetic and cellular determinants of the formation of syncytia may reveal insights into the physiological and pathological consequences of cell-cell fusion.

Functions and applications of artificial intelligence in droplet microfluidics.

Droplet microfluidics has emerged as a powerful technology to perform high-throughput experiments, while artificial intelligence (AI) serves as a functional tool to analyze a large set of multiplex data. Their convergence creates new opportunities in autonomous system optimization and control, enabling various innovative functions and applications. In this study, we elucidate the basic principles of AI and elaborate on its main functions. The intelligent microfluidic systems applied in droplet generation, material synthesis, and biological analysis are summarized, with their working mechanisms and enabled new functions highlighted. Moreover, we elucidate current challenges in a more widespread combination of AI and droplet microfluidics and offer our perspectives on potential strategies to tackle these challenges. We hope that this review can deepen our understanding of intelligent droplet microfluidics and inspire more functional designs tailored to emerging demands.

Self-synchronization of reinjected droplets for high-efficiency droplet pairing and merging.

Droplet merging serves as a powerful tool to add reagents to moving droplets for biological and chemical reactions. However, unsynchronized droplet pairing impedes high-efficiency merging. Here, we develop a microfluidic design for the self-synchronization of reinjected droplets. A periodic increase in the hydrodynamic resistance caused by droplet blocking a T-junction enables automatic pairing of droplets. After inducing spacing, the paired droplets merge downstream under an electric field. The blockage-based design can achieve a 100% synchronization efficiency even when the mismatch rate of droplet frequencies reaches 10%. Over 98% of the droplets can still be synchronized at nonuniform droplet sizes and fluctuating reinjection flow rates. Moreover, the droplet pairing ratio can be adjusted flexibly for on-demand sample addition. Using this system, we merge two groups of droplets encapsulating enzyme/substrate, demonstrating its capacity to conduct multi-step reactions. We also combine droplet sorting and merging to coencapsulate single cells and single beads, providing a basis for high-efficiency single-cell sequencing. We expect that this system can be integrated with other droplet manipulation systems for a broad range of chemical and biological applications.

A composition-tunable cold atmospheric plasma chip for multiplex-treatment of cells.

Cold atmospheric plasma treatment promises a targeted cancer therapy due to its selectivity and specificity in killing tumor cells. However, the current plasma exposure devices produce diverse and coupled reactive species, impeding the investigation of the underlying plasma-anticancer mechanisms. Also, the limited mono-sample and mono-dosage treatment modality result in tedious and manual experimental tasks. Here, we propose a cold atmospheric plasma chip producing targeted species, delivering multiple dosages, and treating multiple cell lines in a single treatment. Three modules are integrated into the chip. The environment control module and multi-inlet gas-feed module coordinately ignite component-tunable and uniformly distributed plasma. The multi-sample holding module enables multiplex treatment: multi-sample and -dosage treatment with single radiation. By exposing the HepG2 cell line to nitrogen-feed plasmas, we prove the crucial role of nitrogen-based species in inhibiting cell growth and stimulating apoptosis. By loading four-type cell lines on our chip, we can identify the most vulnerable cell line for plasma oncotherapy. Simultaneously, three-level treatment dosages are imposed on the cells with single radiation to optimize the applicable treatment dosage for plasma oncotherapy. Our chip will broaden the design principles of plasma exposure devices, potentially help clarify plasma-induced anticancer mechanisms, and guide the clinical application of plasma-based oncotherapy.

High-Throughput Generation, Manipulation, and Degradation of Magnetic Nanoparticle-Laden Alginate Core-Shell Beads for Single Bacteria Culturing Analysis.

Microbes could be found almost everywhere around us and have significant impacts on our human society. The treatment of microorganisms has long been seen as a complex problem. Till now, most of the genetic and phenotypic information regarding rare species is buried in the bulk microbial colony due to a lack of efficient tools to screen live bacteria. Droplet microfluidics offers a powerful approach to address this problem. However, the interactions among bacteria and their living environment are entirely restricted by the water/oil interfaces in conventional water/oil single emulsion-based microfluidic systems. Here, we demonstrate an oil-mediated all-aqueous microfluidic workflow that can overcome this drawback. In contrast to the previous works, our all-aqueous culturing environment allows cell-cell and cell-environment interactions, thus facilitating the growth of bacteria. Fe3O4 magnetic nanoparticles added into the alginate beads enables on-chip manipulation of the microcapsules. The core-shell structure separately encapsulates bacteria and magnetic particles in the core and shell to avoid contamination. We demonstrate the feasibility of this approach by single bacterium culturing in droplet-templated alginate beads. Finally, a new approach is proposed to degrade the alginate beads for post-treatment. This novel microfluidic workflow can create new opportunities for microbial applications, such as bacteria culturing and screening.

Oil-mediated high-throughput generation and sorting of water-in-water droplets.

Aqueous two-phase system (ATPS) droplets have demonstrated superior compatibility over conventional water-in-oil droplets for various biological assays. However, the ultralow interfacial tension hampers efficient and stable droplet generation, limiting further development and more extensive use of such approaches. Here, we present a simple strategy to employ oil as a transient medium for ATPS droplet generation. Two methods based on passive flow focusing and active pico-injection are demonstrated to generate water-water-oil double emulsions, achieving a high generation frequency of ~2.4 kHz. Through evaporation of the oil to break the double emulsions, the aqueous core can be released to form uniform-sized water-in-water droplets. Moreover, this technique can be used to fabricate aqueous microgels, and the introduction of the oil medium enables integration of droplet sorting to produce single-cell-laden hydrogels with a harvest rate of over 90%. We believe that the demonstrated high-throughput generation and sorting of ATPS droplets represent an important tool to advance droplet-based tissue engineering and single-cell analyses.

On-Demand Droplet Collection for Capturing Single Cells.

Droplet-based microfluidic techniques are extensively used in efficient manipulation and genome-wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single-droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on-demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on-demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell-to-cell variations.

Microfluidic Technology for Nucleic Acid Aptamer Evolution and Application.

The intersection of microfluidics and aptamer technologies holds particular promise for rapid progress in a plethora of applications across biomedical science and other areas. Here, the influence of microfluidics on the field of aptamers, from traditional capillary electrophoresis approaches through innovative modern-day approaches using micromagnetic beads and emulsion droplets, is reviewed. Miniaturizing aptamer-based bioassays through microfluidics has the potential to transform diagnostics and embedded biosensing in the coming years.

A Microfluidic System for One-Chip Harvesting of Single-Cell-Laden Hydrogels in Culture Medium.

Single-cell analysis has shown great potential to fully quantify the distribution of cellular behaviors among a population of individuals. Through isolation and preservation of single cells in the aqueous phase, droplet encapsulation followed by gelation enables high-throughput analysis in biocompatible microgels. However, the lack of control over the number of cells encapsulated and complicated gelation processes significantly limit its efficiency. Here, a microfluidic system for one-chip harvesting of single-cell-laden microgels is presented. Through ultraviolet irradiation, an on-chip gelation technique is seamlessly combined with droplet generation to realize high-throughput fabrication of microscale hydrogels in microfluidic channel. Moreover, a sorting module is introduced to simultaneously complete cell-laden microgel selection and transfer into culture medium. To demonstrate the efficiency of this method, two types of single cells are respectively encapsulated and collected, showing desirable single-cell encapsulation and cell viability. This technique realizes integrated droplet gelation, microgel sorting, and transfer into culture medium, allowing high-throughput analysis of single cells and comprehensive understanding of the cellular specificity.

Emerging microfluidic devices for cell lysis: a review.

Intracellular components containing information about genetic and disease characteristics are key substances to clinical diagnostics. Cell lysis is therefore a crucial step for efficient extraction and the subsequent analysis of intracellular components. With the advent of advanced manufacturing techniques, a number of micro systems have been proposed and applied for manipulating cells on chips. In this paper, we review emerging microfluidic devices for cell lysis. Different lysis mechanisms and related techniques are compared. The technical details, advantages, and limitations of various microfluidic devices are discussed.