No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Pinch-off Droplets Generator Using Microscale Gigahertz Acoustics
Abstract
The generation and dispensing of microdroplets is a vital process in various fields such as biomedicine, medical diagnosis and chemistry. However, most methods still require the structures of nozzles or microchannels to assist droplet generation, which leads to limitations on system flexibility and restrictions on the size range of the generated droplets. In this paper, we propose a nozzle-free acoustic-based method for generating droplets using a gigahertz (GHz) bulk acoustic wave (BAW). Unlike most of the acoustofluidic approaches, the proposed method produces the droplet by pinching-off the liquid column generated by the acoustic body force at the oil-water interface. Benefitting from the focused acoustic energy and small footprint of the device, four orders of magnitude (ranging from 2 μm to 1800 μm) of droplet size could be produced by controlling the working time and power of the device. We also demonstrated cell encapsulation in the droplet and a high cell viability was achieved. The proposed acoustic-based droplet generation method exhibits capacity for generating droplets with a wide size range, versatility toward different viscosities, as well as biocompatibility for handling viable samples, which shows potential in miniaturization and scalability.
... In recent years, acoustic waves induced by the piezoelectric effect have been employed to drive liquids and manipulate particles in a wide range of miniaturized systems (especially microfluidic systems) [57][58][59][60][61][62][63][64][65][66][67]. These piezoelectric chips fabricated via advanced microelectromechanical system techniques [68,69] enable acoustofluidic micropumps (AFMPs) with great portability (simple and small structures, low power consumption, and low working voltage), high biocompatibility, a fast response time, and fair extensibility [23,[50][51][52][53][54], which theoretically meets the requirements of a portable system as an ideal micropump. ...
... However, the existing AFMPs have unsatisfactory pumping performance due to their limited energy transduction efficiency (Table S2). Recently, gigahertz (GHz) bulk acoustic waves (BAWs) have been demonstrated to induce intense acoustic streaming in a very small area due to the focused acoustic wave and rapid dissipation at the device-liquid interface, which have been applied for different applications, including solution mixing, droplet dispensing, and bioparticle manipulation [59,62,66,67,70,71]. However, for pumping applications, the GHz acoustic streaming tends to be vortical in nature, which prevents the formation of unidirectional flow. ...
Miniaturization of health care, biomedical, and chemical systems is highly desirable for developing point-of-care testing (POCT) technologies. In system miniaturization, micropumps represent one of the major bottlenecks due to their undesirable pumping performance at such small sizes. Here, we developed a microelectromechanical system fabricated acoustic micropump based on an ultrahigh-frequency bulk acoustic wave resonator. The concept of an inner-boundary-confined acoustic jet was introduced to facilitate unidirectional flow. Benefitting from the high resonant frequency and confined acoustic streaming, the micropump reaches 32.620 kPa/cm³ (pressure/size) and 11.800 ml/min∙cm³ (flow rate/size), showing a 2-order-of-magnitude improvement in the energy transduction efficiency compared with the existing acoustic micropumps. As a proof of concept, the micropump was constructed as a wearable and wirelessly powered integrated drug delivery system with a size of only 9×9×9 mm³ and a weight of 1.16 g. It was demonstrated for ocular disease treatment through animal experimentation and a human pilot test. With superior pumping performance, miniaturized pump size, ultralow power consumption, and complementary metal–oxide–semiconductor compatibility, we expect it to be readily applied to various POCT applications including clinical diagnosis, prognosis, and drug delivery systems.
... For airborne scenario, the reported systems typically work in the frequency of 40 kHz on standing acoustic waves with wavelength about 8.5 mm [29,30] (basically following the design prototyped by Drinkwater [40] in 2017). Airborne BAW based systems can manipulate expanded polystyrene (EPS) particles or liquid droplets, and have been successfully applied for airborne droplet printing [41], highlighting the excellent selectivity due to the dynamic acoustic field modulation capability. However, airborne BAW based systems are very vulnerable to environmental disturbances like air flow or solid obstacles, thus have rarely been reported in precision particle manipulation reaching micron scale. ...
The noncontact acoustic manipulation of particles, bio-samples, droplets, and air bubbles has emerged as a promising technology in fields of biology, chemistry, medicine, etc. The noncontact nature offers significant advantages in terms of bio-compatibility, contamination-free, and material versatility. However, current noncontact acoustic manipulation techniques still lack adequate selectivity, robustness, and precision controllability in complex environments. To this end, this paper proposes an automated noncontact manipulation system that leverages a high-density ultrasonic phased transducer array in combination with a microscope to further optimize and enhance the controllability and flexibility of noncontact particle manipulation. This work presents several notable contributions. First, we successfully realized selective particle manipulation, allowing instantaneous interaction with users to perform user-designated and objective-oriented manipulation tasks. Second, we integrated closed-loop control strategy into the system that effectively mitigates misalignment errors induced by the trapping stiffness heterogeneity of acoustic trap, and enables automated precision position control of particles in complex environments (in 30 mm wide workspace, positioning precision is one-fortieth of the wavelength). Third, we proposed a reconfigurable acoustic trap design method, named pseudo-vortex trap, featuring real-time computing and trapping particles larger than the wavelength. The system setup, the calibration specifics, the acoustic trap design methodology, and the corresponding visual servo control scheme (in terms of selective trapping, precision positioning, and dynamic trajectory planning) are given in detail in the paper. Meanwhile, the trapping stiffness and the manipulation stability is also analyzed in this work. Experimental results well demonstrated the effectiveness of proposed system.
... not suitable for rapid adaptation and use by novice researchers due to the licensed capillary size requirements and post-processing needed to generate pico-scale droplets. Other researchers have utilized microelectromechanical systems (MEMS) to generate microdroplets [20][21][22] , demonstrating their ability to consistently and precisely produce droplets. However, the size of the generated droplets tends to be relatively large, typically exceeding 1 nL. ...
Picolitre monodisperse droplet printing technology has important applications in biochemistry, such as accounting for quantitative analysis and single-cell analysis, and can be used for parallel high-throughput analysis of biomarkers and chemicals. However, commonly used droplet generation devices require complex control systems or customised microfluidic chips, making them costly and difficult for researchers to operate. Additionally, generating picolitre monodisperse droplets with microfluidic devices necessitates the introduction of an oil phase to block and separate the liquid. This requirement can reduce the throughput of the target droplets and cause cell contamination, hindering the adoption of this technology. By employing a common 1-mm-diameter capillary in the laboratory in combination with a piezoelectric transducer, we have achieved on-demand picolitre droplet printing of less than 100 pL in an oil-free environment. The device was found to be biocompatible with K562 cells. This approach is less costly, offers greater operational freedom, and is easier to integrate with other downstream assay modules or even handheld cell-printing devices. This study holds great potential for application in areas such as single-cell analysis, cell sampling, and pharmaceutical analysis.
Droplet microarrays (DMAs) leveraging wettability differences are instrumental in digital immunoassays, single-cell analysis, and high-throughput screening. This study introduces an enhanced Teflon lift-off process to fabricate hydrophilic-hydrophobic patterns on a...
Manipulating objects with acoustics has been developed for hundreds of years since the Chladni pattern in the gaseous environment was exhibited. In recent decades, acoustic manipulation in microfluidics, known as...
On‐demand controlled drug delivery is essential for the treatment of a wide range of chronic diseases. As the drug is released at the time when required, its efficacy is boosted and the side effects are minimized. However, so far, drug delivery devices often rely on the passive diffusion process for a sustained release, which is slow and uncontrollable. Herein, a miniaturized microfluidic device for wirelessly controlled ultrafast active drug delivery is presented, driven by an oscillating solid–liquid interface. The oscillation generates acoustic streaming in the drug reservoir, which opens an elastic valve to deliver the drug. High‐speed microscopy reveals the fast response of the valve on the order of 1 ms, which is more than three orders of magnitude faster than the start‐of‐the‐art. The amount of the released drug exhibits a linear relationship with the working time and the electric power applied to the ultrasonic resonator. The trigger of the release is wirelessly controlled via a magnetic field, and the system shows stable output in a continuous experiment for two weeks. The integrated system shows great promise as a long‐term controlled drug delivery implant for chronic diseases.
Over the past two decades, digital microfluidic biochips have been in much demand for safety-critical and biomedical applications and increasingly important in point-of-care analysis, drug discovery, and immunoassays, among other areas. However, for complex bioassays, finding routes for the transportation of droplets in an electrowetting-on-dielectric digital biochip while maintaining their discreteness is a challenging task. In this study, we propose a deep reinforcement learning-based droplet routing technique for digital microfluidic biochips. The technique is implemented on a distributed architecture to optimize the possible paths for predefined source–target pairs of droplets. The actors of the technique calculate the possible routes of the source–target pairs and store the experience in a replay buffer, and the learner fetches the experiences and updates the routing paths. The proposed algorithm was applied to benchmark suites I and III as two different test benches, and it achieved significant improvements over state-of-the-art techniques.
Superhydrophilic–superhydrophobic patterned surfaces constitute a branch of surface chemistry involving the two extreme states of superhydrophilicity and superhydrophobicity combined on the same surface in precise patterns. Such surfaces have many advantages, including controllable wettability, enrichment ability, accessibility, and the ability to manipulate and pattern water droplets, and they offer new functionalities and possibilities for a wide variety of emerging applications, such as microarrays, biomedical assays, microfluidics, and environmental protection. This review presents the basic theory, simplified fabrication, and emerging applications of superhydrophilic–superhydrophobic patterned surfaces. First, the fundamental theories of wettability that explain the spreading of a droplet on a solid surface are described. Then, the fabrication methods for preparing superhydrophilic–superhydrophobic patterned surfaces are introduced, and the emerging applications of such surfaces that are currently being explored are highlighted. Finally, the remaining challenges of constructing such surfaces and future applications that would benefit from their use are discussed.
Digital bioassays based on single-molecule enzyme reactions represent a new class of bioanalytical methods that enable the highly sensitive detection of biomolecules in a quantitative manner. Since the first reports of these methods in the 2000s, there has been significant growth in this new bioanalytical strategy. The principal strategy of this method is to compartmentalize target molecules in micron-sized reactors at the single-molecule level and count the number of microreactors showing positive signals originating from the target molecule. A representative application of digital bioassay is the digital enzyme-linked immunosorbent assay (ELISA). Owing to their versatility, various types of digital ELISAs have been actively developed. In addition, some disease markers and viruses possess catalytic activity, and digital bioassays for such enzymes and viruses have, thus, been developed. Currently, with the emergence of new microreactor technologies, the targets of this methodology are expanding from simple enzymes to more complex systems, such as membrane transporters and cell-free gene expression. In addition, multiplex or multiparametric digital bioassays have been developed to assess precisely the heterogeneities in sample molecules/systems that are obscured by ensemble measurements. In this review, we first introduce the basic concepts of digital bioassays and introduce a range of digital bioassays. Finally, we discuss the perspectives of new classes of digital bioassays and emerging fields based on digital bioassay technology.
A continuous flow cascade of multi-step catalytic reactions is a cutting-edge concept to revolutionize stepwise catalytic synthesis yet is still challenging in practical applications. Herein, a method for practical one-pot cascade catalysis is developed by combining Pickering emulsions with continuous flow. Our method involves co-localization of different catalytically active sub-compartments within droplets of a Pickering emulsion yielding cell-like microreactors, which can be packed in a column reactor for continuous flow cascade catalysis. As exemplified by two chemo-enzymatic cascade reactions for the synthesis of chiral cyanohydrins and chiral ester, 5 − 420 fold enhancement in the catalysis efficiency and as high as 99% enantioselectivity were obtained even over a period of 80 − 240 h. The compartmentalization effect and enriching-reactant properties arising from the biomimetic microreactor are theoretically and experimentally identified as the key factors for boosting the catalysis efficiency and for regulating the kinetics of cascade catalysis.
The oral administration of therapeutic peptides and proteins is favoured from a patient and commercial point of view. In order to reach the systemic circulation after oral administration, these drugs have to overcome numerous barriers including the enzymatic, sulfhydryl, mucus and epithelial barrier. The development of oral formulations for therapeutic peptides and proteins is therefore challenging. Among the most promising formulation approaches are lipid-based nanocarriers such as oil-in-water nanoemulsions, self-emulsifying drug delivery systems (SEDDS), solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), liposomes and micelles. As the lipophilic character of therapeutic peptides and proteins can be tremendously increased such as by the formation of hydrophobic ion pairs (HIP) with hydrophobic counter ions, they can be incorporated in the lipophilic phase of these carriers. Since gastrointestinal (GI) peptidases as well as sulfhydryl compounds such as glutathione and dietary proteins are too hydrophilic to enter the lipophilic phase of these carriers, the incorporated therapeutic peptide or protein is protected towards enzymatic degradation as well as unintended thiol/disulfide exchange reactions. Stability of lipid-based nanocarriers towards lipases can be provided by the use to excipients that are not or just poorly degraded by these enzymes. Nanocarriers with a size < 200 nm and a mucoinert surface such as PEG or zwitterionic surfaces exhibit high mucus permeating properties. Having reached the underlying absorption membrane, lipid-based nanocarriers enable paracellular and lymphatic drug uptake, induce endocytosis and transcytosis or simply fuse with the cell membrane releasing their payload into the systemic circulation. Numerous in vivo studies provide evidence for the potential of these delivery systems. Within this review we provide an overview about the different barriers for oral peptide and protein delivery, highlight the progress made on lipid-based nanocarriers in order to overcome them and discuss strengths and weaknesses of these delivery systems in comparison to other technologies.
We 3D print micro-robotic swimmers with the size of animal cells using a Nanoscribe. The micro-swimmers are powered by the microstreaming flows induced by the oscillating air bubbles entrapped within the micro-robotic swimmers. Previously, micro-swimmers propelled by acoustic streaming require the use of a magnetic field or an additional ultrasound transducer to steer their direction. Here, we show a two-bubble based micro-swimmer that can be propelled and steered entirely using one ultrasound transducer. The swimmer displays boundary following traits similar to those biological swimmers that are known to be important for performing robust biological functions. The micro-robotic swimmer has the potential to advance the current technology in targeted drug delivery and remote microsurgery.
Digital bioassays are powerful methods to detect rare analytes from complex mixtures and study the temporal processes of individual entities within biological systems. In digital bioassays, a crucial first step is the discretization of samples into a large number of identical independent partitions. Here, we developed a rapid and facile sample partitioning method for versatile digital bioassays. This method is based on a detachable self-digitization (DSD) chip which couples a reversible assembly configuration and a predegassing-based self-pumping mechanism to achieve an easy, fast, and large-scale sample partitioning. The DSD chip consists of a channel layer used for loading the sample and a microwell layer used for holding the sample partitions. Benefitting from its detachability, the chip avoids a lengthy oil flushing process used to remove the excess sample in loading channels and can compartmentalize a sample into more than 100,000 wells of picoliter volume with densities up to 14,000 wells/cm² in less than 30 s. We also demonstrated the utility of the proposed method by applying it to digital PCR and digital microbial assays.
Direct full‐color image photodetectors without dichroic prisms or sophisticated color filters have considerable advantages in target recognition and information acquisition for electronic eyes and wearable sensors. However, the ability to combine various multispectral semiconductors in a high‐resolution and cost‐effective manner is still challenging. Here, high‐resolution electrohydrodynamic (EHD) printing, together with ionic liquid methylammonium acetate (MAAc) as the solvent, is first introduced to directly integrate various spectral‐response perovskite films into a pixelized full‐color photodetector. EHD printing enables micro/nanopatterning by using high electrical force to induce jetting, and MAAc improves film quality with scant pinholes and large‐size grains by decreasing the perovskite growth rate. By optimizing the printing process and crystallization condition, 1 µm perovskite dot arrays are EHD printed; this is, to the best of knowledge, the smallest printed feature size of perovskite application. And the photodetector still achieves high R and D* values of 14.97 A W‐1 and 1.41 × 1012 Jones, respectively. Finally, an integrated flexible full‐color image photodetector is constructed, which successfully realizes light signal detection and color recognition, paving a versatile and competitive approach for future full‐color image sensors and artificial vision systems. Direct full‐color image photodetectors have considerable advantages in electronic eyes and wearable sensors. Here, electrohydrodynamic (EHD) printing of an ionic‐liquid‐based ink is first introduced to realize 1 µm perovskite dot arrays, high‐performance photodetectors (R and D* values are 14.97 A W–1 and 1.41 × 1012 Jones, respectively), and flexible pixelized full‐color image sensors, proving the superiority of EHD printing in perovskite optoelectronics fabrication.
Hydrodynamic force loading platforms for controllable cell mechanical deformation play an essential role in modern cell technologies. Current systems require assistance from specific microstructures thus limiting the controllability and flexibility in cell shape modulation, and studies on real‐time 3D cell morphology analysis are still absent. This article presents a novel platform based on acoustic streaming generated from a gigahertz device for cell shape control and real‐time cell deformation analysis. Details in cell deformation and the restoration process are thoroughly studied on the platform, and cell behavior control at the microscale is successfully achieved by tuning the treating time, intensity, and wave form of the streaming. The application of this platform in cell membrane permeability modulation and analysis is also exploited. Based on the membrane reorganization during cell deformation, the effects of deformation extent and deformation patterns on membrane permeability to micro‐ and macromolecules are revealed. This technology has shown its unique superiorities in cell mechanical manipulation such as high flexibility, high accuracy, and pure fluid force operation, indicating its promising prospect as a reliable tool for cell property study and drug therapy development.
We introduced and validated a drop-on-demand method to print cells. The method uses low energy nanosecond laser (wavelength: 532 nm) pulses to generate a transient microbubble at the distal end of a glass microcapillary supplied with bio-ink. Microbubble expansion results in the ejection of a cell-containing micro-jet perpendicular to the irradiation axis, a method we coined Laser Induced Side Transfer (LIST). We show that the size of the deposited bio-ink droplets can be adjusted between 165 and 325 µm by varying the laser energy. We studied the corresponding jet ejection dynamics and determined optimal conditions for satellite droplet-free bioprinting. We demonstrated droplet bio-printing up to a 30 Hz repetition rate, corresponding to the maximum repetition rate of the used laser. Jet ejection dynamics indicate that LIST can potentially reach 2.5 kHz. Finally, we show that LIST-printed human umbilical vein endothelial cells (HUVECs) present negligible loss of viability and maintain their abilities to migrate, proliferate and form intercellular junctions. Sample preparation is uncomplicated in LIST, while with further development bio-ink multiplexing can be attained. LIST could be widely adapted for applications requiring multiscale bioprinting capabilities, such as the development of 3D drug screening models and artificial tissues.
Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility. Millions of primary IgG-secreting cells from mouse and human are characterized for activity and antibody sequence at the single-cell level.
Droplet microfluidics has revolutionized the study of single cells. The ability to compartmentalize cells within picoliter droplets in microfluidic devices has opened up a wide range of strategies to extract information at the genomic, transcriptomic, proteomic, or metabolomic level from large numbers of individual cells. Studying the different molecular landscapes at single‐cell resolution has provided the authors with a detailed picture of intracellular heterogeneity and the resulting changes in cellular phenotypes. In addition, these technologies have aided in the discovery of rare cells in tumors or in the immune system, and left the authors with a deeper understanding of the fundamental biological processes that determine cell fate. This review aims to provide a detailed overview of the various droplet microfluidic strategies reported in the literature, taking into account the sometimes subtle differences in workflow or reagents that enable or improve certain protocols. Specifically, approaches to targeted‐ and whole‐genome analysis, as well as whole‐transcriptome profiling techniques, are reviewed. In addition, an up‐to‐date overview of new methods to characterize and quantify single‐cell protein levels, and of developments to screen secreted molecules such as antibodies, cytokines, or metabolites at the single‐cell level, is provided.
Fabrication of artificial biomimetic materials has attracted abundant attention. As one of the subcategories of biomimetic materials, artificial cells are highly significant for multiple disciplines and their synthesis has been intensively pursued. In order to manufacture robust “alive” artificial cells with high throughput, easy operation, and precise control, flexible microfluidic techniques are widely utilized. Herein, recent advances in microfluidic‐based methods for the synthesis of droplets, vesicles, and artificial cells are summarized. First, the advances of droplet fabrication and manipulation on the T‐junction, flow‐focusing, and coflowing microfluidic devices are discussed. Then, the formation of unicompartmental and multicompartmental vesicles based on microfluidics are summarized. Furthermore, the engineering of droplet‐based and vesicle‐based artificial cells by microfluidics is also reviewed. Moreover, the artificial cells applied for imitating cell behavior and acting as bioreactors for synthetic biology are highlighted. Finally, the current challenges and future trends in microfluidic‐based artificial cells are discussed. This review should be helpful for researchers in the fields of microfluidics, biomaterial fabrication, and synthetic biology.
Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (105 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named ‘droplet single-cell assay for transposase-accessible chromatin using sequencing’ (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform. Combining microfluidics with combinatorial indexing enables rapid mapping of genome accessibility in hundreds of thousands of single cells.
On-demand droplet dispensing systems are indispensable tools in bioanalytical fields, such as microarray
fabrication. Biomaterial solutions can be very limited and expensive, so minimizing the use of solution per
spot produced is highly desirable. Here, we proposed a novel droplet dispensing method which utilizes a
gigahertz (GHz) acoustic resonator to deposit well-defined droplets on-demand. This ultra-high frequency
acoustic resonator induces a highly localized and strong body force at the solid–liquid interface, which
pushes the liquid to generate a stable and sharp “liquid needle” and further delivers droplets to the target
substrate surface by transient contact. This approach is between contact and non-contact methods, thus
avoiding some issues of traditional methods (such as nozzle clogging or satellite spots). We demonstrated
the feasibility of this approach by fabricating high quality DNA and protein microarrays on glass and flexible
substrates. Notably, the spot size can be delicately controlled down to a few microns (femtoliter in volume). Because of the CMOS compatibility, we expect this technique to be readily applied to advanced biofabrication processes.
Dispensing and manipulation of small droplets is important in bioassays, chemical analysis and patterning of functional inks. So far, dispensing of small droplets has been achieved by squeezing the liquid out of a small orifice similar in size to the droplets. Here we report that instead of squeezing the liquid out, small droplets can also be dispensed advantageously from large orifices by draining the liquid out of a drop suspended from a nozzle. The droplet volume is adjustable from attolitre to microlitre. More importantly, the method can handle suspensions and liquids with viscosities as high as thousands mPa s markedly increasing the range of applicable liquids for controlled dispensing. Furthermore, the movement of the dispensed droplets is controllable by the direction and the strength of an electric field potentially allowing the use of the droplet for extracting analytes from small sample volume or placing a droplet onto a pre-patterned surface.
We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in suspension into a single aggregate in each well of the microplate and by this nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy, and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to follow the cell culture from production to analysis of 3D tissue or tumor models by the use of microscopy in one single microplate device.
Microscopic water-in-oil droplets are a versatile chemical and biological platform whose dimensions result in short reaction times and require minuscule amounts of reagent. Methods exist for the production of droplets, though the vast majority are only able to do so in continuous flows, restricting the ability to independently control reactions of individual droplets, a prerequisite for programmable digital microfluidics. Here we present a novel method to produce individual picoliter-scale droplets on-demand using surface acoustic waves (SAW). Acoustic forces arising from SAW act on the oil-water interface, creating a droplet whose volume is defined by the applied power, duration of the force and system geometry. Additionally, this method is able to pre-concentrate particles simultaneously with droplet production, meaning that particles and cells, even if in a dilute mixture, can be easily encapsulated. Our method is expected to be applicable to high-throughput screening, bioreactor creation and other microfluidic processes.
Recently, there has been growing interest in applying bioprinting techniques to stem cell research. Several bioprinting methods have been developed utilizing acoustics, piezoelectricity, and lasers to deposit living cells onto receiving substrates. Using these technologies, spatially defined gradients of immobilized biomolecules can be engineered to direct stem cell differentiation into multiple subpopulations of different lineages. Stem cells can also be patterned in a high-throughput manner onto flexible implementation patches for tissue regeneration or onto substrates with the goal of accessing encapsulated stem cells of interest for genomic analysis. Here, we review recent achievements with bioprinting technologies in stem cell research, and identify future challenges and potential applications including tissue engineering and regenerative medicine, wound healing, and genomics.
On-demand droplet generation from a continuously flowing stream of aqueous phase has profound applications in dropletbased microfluidics for rare event encapsulation studies. Here, we present acoustic relocation-based droplet generation from co-flowing immiscible fluids in an on-demand manner using bulk acoustic wave (BAW). After on-demand droplet generation, droplets are isolated using the same acoustic force resulted from BAW. Two different acoustic relocation regimes are observed, namely, stream to droplet relocation and stream to stream relocation regime. Our experimental observation reveals that to generate droplets from co-flowing fluids, the following conditions must be satisfied. First, the co-flowing immiscible stream should be maintained in acoustic relocation conditions (Cac > 1); Second, the capillary instability should be triggered during the relocation process, which happens at capillary numbers of the co-flowing fluids should be less than 0.2 (CaL and CaH < 0.2). Finally, using BAW microfluidic chip, droplets containing microparticle were produced ondemand from co-flowing streams wherein the microparticles are added in one of the phases.KeywordsBulk acoustic waveEncapsulationAcoustic relocationCo-flowCapillary instabilityOn-demand droplet
Over the past few decades, acoustofluidics, one of the branches of microfluidics, has rapidly developed as a multidisciplinary cutting edge research topic, covering many biomedical and bioanalytical applications. Acoustofluidics usually utilizes acoustic pressure and acoustic streaming effects to manipulate liquids and bioparticles. Acoustic manipulation using acoustic radiation force has been widely studied; however, with the recent development of new piezoelectric devices that enable faster acoustic streaming, particle manipulations using drag force induced by acoustic streaming have attracted more attention. Despite many review articles on acoustic radiation force-based acoustophoresis, acoustic streaming is less frequently covered. Here, we review the recent development of microscale acoustic streaming, especially high-frequency transducer-induced high-speed streaming, confinement and programed streaming, and acoustic streaming tweezers, which combine the acoustic radiation force and drag force to tackle the size limitations of conventional acoustic manipulations. A brief review of acoustic streaming theory and its generation is summarized. Recent progress in applying acoustic streaming for fluidic handling and bioparticle manipulations is reviewed. Representative applications of micro acoustic streaming are provided, and the key issues in these applications are analyzed. Finally, the future prospects of micro acoustic streaming in bioanalytical and biomedical applications are discussed.
The hydrodynamic method mimics the in vivo environment of the mechanical effect on cell stimulation, which not only modulates cell physiology but also shows excellent intracellular delivery ability. Herein, a hydrodynamic intracellular delivery system based on the gigahertz acoustic streaming (AS) effect is proposed, which presents powerful targeted delivery capabilities with high efficiency and universality. Results indicate that the range of cells with AuNR introduction is related to that of AS, enabling a tunable delivery range due to the adjustability of the AS radius. Moreover, with the assistance of AS, the organelle localization delivery of AuNRs with different modifications is enhanced. AuNRs@RGD is inclined to accumulate in the nucleus, while AuNRs@BSA tend to enter the mitochondria and AuNRs@PEGnK tend to accumulate in the lysosome. Finally, the photothermal effect is proved based on the large quantities of AuNRs introduced via AS. The abundant introduction of AuNRs under the action of AS can achieve rapid cell heating with the irradiation of a 785 nm laser, which has great potential in shortening the treatment cycle of photothermal therapy (PTT). Thereby, an efficient hydrodynamic technology in AuNR introduction based on AS has been demonstrated. The outstanding location delivery and organelle targeting of this method provides a new idea for precise medical treatment.
Acoustic fields are ideal for micromanipulation, being biocompatible and with force gradients approaching the scale of single cells. They have accordingly found use in a variety of microfluidic devices, including for microscale patterning, separation, and mixing. The bulk of work in acoustofluidics has been predicated on the formation of standing waves that form periodic nodal positions along which suspended particles and cells are aligned. An evolving range of applications, however, requires more targeted micromanipulation to create unique patterns and effects. To this end, recent work has made important advances in improving the flexibility with which acoustic fields can be applied, impressively demonstrating generating arbitrary arrangements of pressure fields, spatially localizing acoustic fields and selectively translating individual particles in ways that are not achievable via traditional approaches. In this critical review we categorize and examine these advances, each of which open the door to a wide range of applications in which single-cell fidelity and flexible micromanipulation are advantageous, including for tissue engineering, diagnostic devices, high-throughput sorting and microfabrication.
The dynamic control of the chemical concentration within droplets is required in numerous droplet microfluidic applications. Here, we propose an acoustofluidic method for the generation of a library of aqueous droplets with the desired chemical concentrations in a continuous oil phase. Surface acoustic waves produced by a focused interdigital transducer interact with two parallel laminar streams with different chemical compositions. Coupling the acoustic waves with the flow streams results in the controlled acoustofluidic mixing of the aqueous solutions through the formation of acoustic streaming flow-induced microvortices. The mixed streams are split at a bifurcation, and one of the streams with a precisely controlled chemical concentration is fed into a T-junction to produce droplets with tunable chemical concentrations. The periodic acoustofluidic mixing of the aqueous streams enables the generation of a droplet library with a well-defined inter-droplet concentration gradient. The proposed method is a promising tool for the on-chip dynamic control of in-droplet chemical concentrations and for next-generation droplet microfluidic applications.
Laser-induced forward transfer (LIFT), unlike inkjet printing, presents few constrains concerning ink viscosity or loading particle size. This is clearly favorable for printed electronics applications, since high solid content inks, such those of screen printing, can be thus transferred in a digital fashion. In this work we propose a study of the transfer mechanisms during the LIFT of a commercially-available silver screen printing ink.
The printing of single voxels on glass through the variation of pulse energy and donor-receiver gap reveals a linear dependence of voxel volume respect pulse energy for low energies and small gaps. The analysis of the transfer dynamics demonstrates that for the entire range of analyzed conditions the deposit takes place through bubble contact with the receiver.
The printing of lines through variation of the overlap between successive voxels reveals that under none of the analyzed conditions we obtain uniform continuous lines through single scan: the lines always show scalloping, bulging, or discontinuities. These defects are a consequence of the modification of the donor film morphology induced by previous pulses in the line, which makes the transfer dynamics unstable. A final multiple scan approach proves the feasibility of the technique for printing uniform stable lines.
Droplet-based microreactors are of great interest to researchers due to their incredible ability in the synthesis of micro/nano-materials with multi-function and complex geometry. In recent years, a broad range of micro/nano-materials has been synthesized in droplet-based microreactors, which provide apparent advantages, such as better reproducibility, reliable automation, and accurate manipulation. In this review, we give a comprehensive and in-depth insight into droplet-based microreactors, covering fundamental research from droplet generation and manipulation to the applications of droplet-based microreactors in micro/nano-material generation. We also explore the outlook for droplet-based microreactors and challenges that lie ahead and give a possible effort direction. We hope this review will promote communications among researchers and entrepreneurs. This article is protected by copyright. All rights reserved.
Microfluidic platforms have changed the paradigm of biochemical experimentation over the past three decades. Prominent within this technology set are droplet-based microfluidic systems, in which passive microfluidic structures are used to rapidly generate and manipulate sub-nanoliter volumes droplets within microchannel environments. Droplets are formed in a continuous and robust fashion through the extrusion and shearing of two mutually immiscible phases in a microchannel, with droplet volumes being precisely controlled through the variation of flow rate ratios and channel dimensions. The rapid production (and analysis) of droplets allows for exceptionally high-throughput experimentation and data acquisition, and configurable channel designs, coupled with on-demand control architectures, engender a range of robust manipulations, such as reagent dosing, droplet fusion, droplet splitting, washing, payload heating, incubation, content dilution and droplet sorting. Accordingly, and unsurprisingly, droplet-based microfluidic systems have become an indispensable and embedded tool within contemporary chemical and biological science.
Mimicking the hierarchical microarchitectures of native myocardium in vitro plays an important role in cardiac tissue engineering. Here we present a novel strategy to produce multiscale conductive scaffolds with layer-specific fiber orientations for cardiac regeneration by combining solution-based and melt-based electrohydrodynamic (EHD) printing techniques. Polycaprolactone (PCL) microfibers were printed by melt-based EHD printing and the fiber orientation was flexibly controlled in a layer-by-layer manner according to user-specific design. The as-printed microfibrous scaffolds can provide the seeded cells necessary contact cues to guide layer-specific cellular alignments. Sub-microscale conductive fibers were simultaneously incorporated inside the well-organized PCL scaffolds by solution-based EHD printing, which significantly improved the conductivity as well as the cellular adhesion and proliferation capacity. The multiscale conductive scaffolds can further direct the multiple-layer alignments of primary cardiomyocytes and facilitate cardiomyocyte-specific gene expressions, which exhibited enhanced synchronous beating behavior compared with pure microfibrous scaffolds. It is envisioned that the proposed hybrid EHD printing technique might provide a promising strategy to fabricate multifunctional micro/nanofibrous scaffolds with biomimetic architectures, electrical conductivity and even biosensing properties for the regeneration of electroactive tissues.
Hypothesis
In this manuscript we examine the stability of an evaporating-unbounded axisymmetric liquid bridge confined between parallel-planar similar or chemically different substrates using both theory and experiments. With a quasistatic assumption we use hydrostatics to estimate the minimum stable volume Vmin via the Young-Laplace equation for Bond numbers 0⩽Bo⩽1, and top/bottom wall contact angles 5°<θ<175° although the primary focus is on wetting and partial wetting fluids. Solving the Young-Laplace equation requires knowledge of appropriate capillary pressure values, which appear as a constant, and may not provide unique solution. To examine uniqueness of numerical solutions and volume minima determined from the Young-Laplace equation for unbounded-axisymmetric liquid bridges we analyzed capillary pressure for large and small liquid volume-asymptotic limits at zero Bond number.
Experiments
Experiments were performed to compare with the volume minima calculations for Bond numbers 0.04⩽Bo⩽0.65. Three substrates of varying surface energy were used, with purified water as the primary liquid. Volume estimates and contact angle data were extracted via image analysis and evaporation rates measured from this data are reported.
Findings
Volume minima were in the range 0.1<Vmin<20 μl depending on Bond number. There was good agreement when comparing predicted volume minima and those determined from experiments for the range of parameters studied.
Lipid droplets are storage organelles at the centre of lipid and energy homeostasis. They have a unique architecture consisting of a hydrophobic core of neutral lipids, which is enclosed by a phospholipid monolayer that is decorated by a specific set of proteins. Originating from the endoplasmic reticulum, lipid droplets can associate with most other cellular organelles through membrane contact sites. It is becoming apparent that these contacts between lipid droplets and other organelles are highly dynamic and coupled to the cycles of lipid droplet expansion and shrinkage. Importantly, lipid droplet biogenesis and degradation, as well as their interactions with other organelles, are tightly coupled to cellular metabolism and are critical to buffer the levels of toxic lipid species. Thus, lipid droplets facilitate the coordination and communication between different organelles and act as vital hubs of cellular metabolism.
Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.
Droplet microfluidics enables cellular encapsulation for biomedical applications such as single-cell analysis, which is an important tool used by biologists to study cells on a single-cell level, and understand cellular heterogeneity in cell populations. However, most cell-encapsulation strategies in microfluidics rely on random encapsulation processes, resulting in large numbers of empty droplets. Therefore, post-sorting of droplets is necessary to obtain samples of purely cell-encapsulating droplets. With the recent advent of aqueous two-phase systems (ATPS) as a biocompatible alternative of the conventional water-in-oil droplet systems for cellular encapsulation, there has also been a focus on integrating ATPS with droplet microfluidics. In this paper, we describe a new technique that combines ATPS-based water-in-water droplets with diamagnetic manipulation to isolate single-cell encapsulating water-in-water droplets, and achieve a purity of 100 % in a single pass. We exploit the selective partitioning of ferrofluid in an ATPS of polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG) and dextran (DEX), to achieve diamagnetic manipulation of water-in-water droplets. A cell-triggered Rayleigh-Plateau instability in the dispersed phase thread results in a size distinction between the cell-encapsulating and empty droplets, enabling diamagnetic separation and sorting of the cell-encapsulating droplets from empty droplets. This is a simple and biocompatible all-aqueous platform for single-cell encapsulation and droplet manipulation, with applications in single-cell analysis.
We report the nonlinear acoustic streaming effect and the fast manipulation of microparticles by microelectromechanical Lamb-wave resonators in a microliter droplet. The device, consisting of four Lamb-wave resonators on a silicon die, generates cylindrical traveling waves in a liquid and efficiently drives nine horizontal vortices within a 1−μl droplet; the performance of the device coincides with the numerical model prediction. Experimentally, the particles are enriched at the stagnation center of the main vortex on the free surface of the droplet in open space without microfluidic channels. In addition, the trajectories of the particles in the droplet can be controlled by the excitation power.
Micro/nano scale biosensors integrated with the local adsorption mask have been demonstrated to have a better limit of detection (LOD) and less sample consumptions. However, the molecular diffusions and binding kinetics in such confined droplet have been less studied which limited further development and application of the local adsorption method and imposed restrictions on discovery of new signal amplification strategies. In this work, we studied the kinetic issues via experimental investigations and theoretical analysis on microfabricated biosensors. Mass sensitive film bulk acoustic resonator (FBAR) sensors with hydrophobic Teflon film covering the non-sensing area as the mask were introduced. The fabricated masking sensors were characterized with physical adsorption of bovine serum albumin (BSA) and specific binding of antibody and antigen. Over an order of magnitude improvement on LOD was experimentally monitored. An analytical model was introduced to discuss the target molecule diffusion and binding kinetics in droplet environment, especially the crucial effects of incubation time, which has been less covered in previous local adsorption related literatures. An incubation time accumulated signal amplification effect was theoretically predicted, experimentally monitored and carefully explained. In addition, device optimization was explored based on the analytical model to fully utilize the merits of local adsorption. The discussions on the kinetic issues are believed to have wide implications for other types of micro/nano fabricated biosensors with potentially improved LOD.
Copyright © 2015 Elsevier B.V. All rights reserved.
It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT.
Copyright © 2015 Elsevier Inc. All rights reserved.
A flow-focusing geometry is integrated into a microfluidic device and used to study drop formation in liquid–liquid systems. A phase diagram illustrating the drop size as a function of flow rates and flow rate ratios of the two liquids includes one regime where drop size is comparable to orifice width and a second regime where drop size is dictated by the diameter of a thin “focused” thread, so drops much smaller than the orifice are formed. Both monodisperse and polydisperse emulsions can be produced.
Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding.
Drop formation at a capillary tip in laminar flow is investigated experimentally. The disperse phase is injected via a needle into another co-flowing immiscible fluid. Two different drop formation mechanisms are distinguished: Either the drops are formed close to the capillary tip—dripping—or they break up from an extended liquid jet—jetting. The effect of the process and material parameters on the drop formation depends on the breakup mechanism and has to be investigated for each flow domain separately. In this study, we focus on dripping. The drop breakup is affected by the flow dynamics of both the disperse and the continuous phase. Consequently, we investigate the effect of flow rates, fluid viscosities and interfacial tension on the droplet size and observe the dynamics of satellite drop generation. Whereas the fundamentals of disperse fluid injection via a capillary into an ambient fluid have been investigated extensively, the focus of this article is on providing a comprehensive experimental data set for proving the applicability of this technique as a dispersing tool. It is shown that drop formation at a capillary tip into a co-flowing ambient liquid represents a promising technique for the production of monodisperse droplets where the droplet size is controlled externally by the flow strength of the continuous phase. The breakup dynamics changes significantly at the transition point from dripping to jetting. Consequently, the transition point between the flow domains represents an important operating point. In this article, dripping is demarcated from jetting by studying the influence of the various material and process parameters on the transition point.
The exploitation of microdroplets produced within microfluidic environments has recently emerged as a new and exciting technological platform for applications within the chemical and biological sciences. Interest in microfluidic systems has been stimulated by a range of fundamental features that accompany system miniaturization. Such features include the ability to process and handle small volumes of fluid, improved analytical performance when compared to macroscale analogues, reduced instrumental footprints, low unit cost, facile integration of functional components and the exploitation of atypical fluid dynamics to control molecules in both time and space. Moreover, microfluidic systems that generate and utilize a stream of sub-nanolitre droplets dispersed within an immiscible continuous phase have the added advantage of allowing ultra-high throughput experimentation and being able to mimic conditions similar to that of a single cell (in terms of volume, pH, and salt concentration) thereby compartmentalizing biological and chemical reactions. This review provides an overview of methods for generating, controlling and manipulating droplets. Furthermore, we discuss key fields of use in which such systems may make a significant impact, with particular emphasis on novel applications in the biological and physical sciences.
This article describes the process of formation of droplets and bubbles in microfluidic T-junction geometries. At low capillary numbers break-up is not dominated by shear stresses: experimental results support the assertion that the dominant contribution to the dynamics of break-up arises from the pressure drop across the emerging droplet or bubble. This pressure drop results from the high resistance to flow of the continuous (carrier) fluid in the thin films that separate the droplet from the walls of the microchannel when the droplet fills almost the entire cross-section of the channel. A simple scaling relation, based on this assertion, predicts the size of droplets and bubbles produced in the T-junctions over a range of rates of flow of the two immiscible phases, the viscosity of the continuous phase, the interfacial tension, and the geometrical dimensions of the device.
Microsystems create new opportunities for the spatial and temporal control of cell growth and stimuli by combining surfaces that mimic complex biochemistries and geometries of the extracellular matrix with microfluidic channels that regulate transport of fluids and soluble factors. Further integration with bioanalytic microsystems results in multifunctional platforms for basic biological insights into cells and tissues, as well as for cell-based sensors with biochemical, biomedical and environmental functions. Highly integrated microdevices show great promise for basic biomedical and pharmaceutical research, and robust and portable point-of-care devices could be used in clinical settings, in both the developed and the developing world.
The manipulation of fluids in channels with dimensions of tens of micrometres--microfluidics--has emerged as a distinct new field. Microfluidics has the potential to influence subject areas from chemical synthesis and biological analysis to optics and information technology. But the field is still at an early stage of development. Even as the basic science and technological demonstrations develop, other problems must be addressed: choosing and focusing on initial applications, and developing strategies to complete the cycle of development, including commercialization. The solutions to these problems will require imagination and ingenuity.
- Tewhey