Robust acoustic trapping and perturbation of single-cell microswimmers illuminate three-dimensional swimming and ciliary coordination.

We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular alga Chlamydomonas reinhardtii is widely used to investigate the mechanisms underlying ciliary beating and coordination. However, freely swimming cells are difficult to image with sufficient resolution to capture cilia motion, necessitating that the cell body be held during experiments. Acoustic confinement is a compelling alternative to use of a micropipette, or to magnetic, electrical, and optical trapping that may modify the cells and affect their behavior. Beyond establishing our approach to studying microswimmers, we demonstrate a unique ability to mechanically perturb cells via rapid acoustic positioning.

Acoustic levitation as a tool for cell-driven self-organization of human cell spheroids during long-term 3D culture.

Acoustic levitation, which allows contactless manipulation of micro-objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell-cell interactions, away from the walls of the chip, which leads to the spontaneous self-organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long-term culture of cells in acoustic levitation is feasible. We also demonstrated that this self-organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium-mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix.

An Acoustofluidic Picoinjector.

Droplet microfluidics has emerged as a valuable technology for a multitude of chemical and biomedical applications, offering the capability to create independent microenvironments for high-throughput assays. Central to numerous droplet microfluidic applications is the picoinjection of materials into individual droplets, yet existing picoinjection methods often exhibit high power requirements, lack biocompatibility, and/or suffer from limited controllability. Here, we present an acoustofluidic picoinjector that generates acoustic pressure at the droplet interface to enable on-demand, energy-efficient, and biocompatible injection at high precision. We validate our platform by performing acid-base titrations by iteratively injecting picoliter volume reagents into droplets to induce pH transitions detectable by color change in solution. Additionally, we demonstrate the versatility of the acoustofluidic picoinjector in the synthesis of metallic nanoparticles, yielding highly monodisperse and reproducible particle morphologies compared to conventional bulk-phase techniques. By facilitating controlled delivery of reagents or biological samples with unparalleled accuracy, acoustofluidic picoinjection broadens the utility of droplet microfluidics for a myriad of applications in chemical and biological research.

Capillary-based, multifunctional manipulation of particles and fluids via focused surface acoustic waves.

Surface acoustic wave (SAW)-enabled acoustofluidic technologies have recently atttracted increasing attention for applications in biology, chemistry, biophysics, and medicine. Most SAW acoustofluidic devices generate acoustic energy which is then transmitted into custom microfabricated polymer-based channels. There are limited studies on delivering this acoustic energy into convenient commercially-available glass tubes for manipulating particles and fluids. Herein, we have constructed a capillary-based SAW acoustofluidic device for multifunctional fluidic and particle manipulation. This device integrates a converging interdigitated transducer to generate focused SAWs on a piezoelectric chip, as well as a glass capillary that transports particles and fluids. To understand the actuation mechanisms underlying this device, we performed finite element simulations by considering piezoelectric, solid mechanic, and pressure acoustic physics. This experimental study shows that the capillary-based SAW acoustofluidic device can perform multiple functions including enriching particles, patterning particles, transporting particles and fluids, as well as generating droplets with controlled sizes. Given the usefulness of these functions, we expect that this acoustofluidic device can be useful in applications such as pharmaceutical manufacturing, biofabrication, and bioanalysis.

Acoustofluidic assembly of primary tumor-derived organotypic cell clusters for rapid evaluation of cancer immunotherapy.

Cancer immunotherapy shows promising potential for treating breast cancer. While patients may have heterogeneous treatment responses for adjuvant therapy, it is challenging to predict an individual patient's response to cancer immunotherapy. Here, we report primary tumor-derived organotypic cell clusters (POCCs) for rapid and reliable evaluation of cancer immunotherapy. By using a label-free, contactless, and highly biocompatible acoustofluidic method, hundreds of cell clusters could be assembled from patient primary breast tumor dissociation within 2 min. Through the incorporation of time-lapse living cell imaging, the POCCs could faithfully recapitulate the cancer-immune interaction dynamics as well as their response to checkpoint inhibitors. Superior to current tumor organoids that usually take more than two weeks to develop, the POCCs can be established and used for evaluation of cancer immunotherapy within 12 h. The POCCs can preserve the cell components from the primary tumor due to the short culture time. Moreover, the POCCs can be assembled with uniform fabricate size and cell composition and served as an open platform for manipulating cell composition and ratio under controlled treatment conditions with a short turnaround time. Thus, we provide a new method to identify potentially immunogenic breast tumors and test immunotherapy, promoting personalized cancer therapy.

Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells.

Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.

A Simulated Investigation of Lithium Niobate Orientation Effects on Standing Acoustic Waves.

The integration of high-frequency acoustic waves with microfluidics has been gaining popularity as a method of separating cells/particles. A standing surface acoustic wave (sSAW) device produces constructive interference of the stationary waves, demonstrating an increase in cell separating efficiency without damaging/altering the cell structure. The performance of an sSAW device depends on the applied input signal, design of the IDT, and piezoelectric properties of the substrate. This work analyzes the characteristics of a validated 3D finite element model (FEM) of LiNbO3 and the effect on the displacement components of the mechanical waves under the influence of sSAWs by considering XY-, YX-, and 1280 YX-cut LiNbO3 with varying electrode length design. We demonstrated that device performance can be enhanced by the interference of multiple waves under a combination of input signals. The results suggest that 1280 YX-cut LiNbO3 is suitable for generating higher-amplitude out-of-plane waves which can improve the effectiveness of acoustofluidics-based cell separation. Additionally, the findings showed that the length of the electrode impacts the formation of the wavefront significantly.

Staged Assembly of Colloids Using DNA and Acoustofluidics.

Assembly of DNA-coated colloids (DNACCs) provides a practical route to programming complex self-assembled materials at the micro/nanoscale. So far, the programmability of DNACC assembly has been extensively exploited internally using different DNA sequences or colloid geometry so that the assembly is mainly manipulated with single-particle spatial resolution such as in crystallization. In this Letter, we present an acoustic approach to externally programming the DNACC assembly with control of spatial resolution over larger scales. We demonstrate assembly of the DNACCs under different acoustic frequencies from stage to stage to produce hierarchical structures that are difficult to fabricate when using DNA coating alone. By programming the acoustic wave frequency, amplitude, and phase, colloidal structures with different morphologies can be assembled. The nonspecific driving force based on acoustic radiation forces at each stage allows our approach to be adopted for most colloidal systems without specific requirements on particle or medium properties.

CeFlowBot: A Biomimetic Flow-Driven Microrobot that Navigates under Magneto-Acoustic Fields.

Aquatic organisms within the Cephalopoda family (e.g., octopuses, squids, cuttlefish) exist that draw the surrounding fluid inside their bodies and expel it in a single jet thrust to swim forward. Like cephalopods, several acoustically powered microsystems share a similar process of fluid expulsion which makes them useful as microfluidic pumps in lab-on-a-chip devices. Herein, an array of acoustically resonant bubbles are employed to mimic this pumping phenomenon inside an untethered microrobot called CeFlowBot. CeFlowBot contains an array of vibrating bubbles that pump fluid through its inner body thereby boosting its propulsion. CeFlowBots are later functionalized with magnetic layers and steered under combined influence of magnetic and acoustic fields. Moreover, acoustic power modulation of CeFlowBots is used to grasp nearby objects and release it in the surrounding workspace. The ability of CeFlowBots to navigate remote environments under magneto-acoustic fields and perform targeted manipulation makes such microrobots useful for clinical applications such as targeted drug delivery. Lastly, an ultrasound imaging system is employed to visualize the motion of CeFlowBots which provides means to deploy such microrobots in hard-to-reach environments inaccessible to optical cameras.

Acoustofluidic platforms for particle manipulation.

There is an increasing interest in acoustics for microfluidic applications. This field, commonly known as acoustofluidics involves the interaction of ultrasonic standing waves with fluids and dispersed microparticles. The combination of microfluidics and the so-called acoustic standing waves (ASWs) led to the development of integrated systems for contact-less on-chip cell and particle manipulation where it is possible to move and spatially localize these particles based on the different acoustophysical properties. While it was initially suggested that the acoustic forces could be harmful to the cells and could impact cell viability, proliferation, or function via phenotypic or even genotypic changes, further studies disproved such claims. This review is summarizing some interesting applications of acoustofluidics in the manipulations of biomaterials, such as cells or subcellular vesicles, in works published mainly within the last 5 years.

Well-free agglomeration and on-demand three-dimensional cell cluster formation using guided surface acoustic waves through a couplant layer.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

Square microchannel enables to focus and orient ellipsoidal Euglena gracilis cells by two-dimensional acoustic standing wave.

Flow cytometry has become an indispensable tool for counting, analyzing, and sorting large cell populations in biological research and medical practice. Unfortunately, it has limitations in the analysis of non-spherically shaped cells due to the variation of their alignment with respect to the flow direction and, hence, the optical interrogation axis, resulting in unreliable cell analysis. Here, we present a simple on-chip acoustofluidic method to fix the orientation of ellipsoidal cells and focus them into a single, aligned stream. Specifically, by generating acoustic standing waves inside a 100 ⋅ 100 µm square-shaped microchannel, we successfully aligned and focused up to 97.7% of a population of Euglena gracilis (an ellipsoidal shaped microalgal species) cells in the center of the microchannel with high precision at a volume rate of 25 to 200 µL min-1. Uniform positioning of ellipsoidal cells is essential for making flow cytometry applicable to the investigation of a greater variety of cell populations and is expected to be beneficial for ecological studies and aquaculture.

Flexible Platform of Acoustofluidics and Metamaterials with Decoupled Resonant Frequencies.

The key challenge for a lab-on-chip (LOC) device is the seamless integration of key elements of biosensing and actuation (e.g., biosampling or microfluidics), which are conventionally realised using different technologies. In this paper, we report a convenient and efficient LOC platform fabricated using an electrode patterned flexible printed circuit board (FPCB) pressed onto a piezoelectric film coated substrate, which can implement multiple functions of both acoustofluidics using surface acoustic waves (SAWs) and sensing functions using electromagnetic metamaterials, based on the same electrode on the FPCB. We explored the actuation capability of the integrated structure by pumping a sessile droplet using SAWs in the radio frequency range. We then investigated the hybrid sensing capability (including both physical and chemical ones) of the structure employing the concept of electromagnetic split-ring resonators (SRRs) in the microwave frequency range. The originality of this sensing work is based on the premise that the proposed structure contains three completely decoupled resonant frequencies for sensing applications and each resonance has been used as a separate physical or a chemical sensor. This feature compliments the acoustofluidic capability and is well-aligned with the goals set for a successful LOC device.

Deterministic Sorting of Submicrometer Particles and Extracellular Vesicles Using a Combined Electric and Acoustic Field.

Sorting of extracellular vesicles has important applications in early stage diagnostics. Current exosome isolation techniques, however, suffer from being costly, having long processing times, and producing low purities. Recent work has shown that active sorting via acoustic and electric fields are useful techniques for microscale separation activities, where combining these has the potential to take advantage of multiple force mechanisms simultaneously. In this work, we demonstrate an approach using both electrical and acoustic forces to manipulate bioparticles and submicrometer particles for deterministic sorting, where we find that the concurrent application of dielectrophoretic (DEP) and acoustophoretic forces decreases the critical diameter at which particles can be separated. We subsequently utilize this approach to sort subpopulations of extracellular vesicles, specifically exosomes (<200 nm) and microvesicles (>300 nm). Using our combined acoustic/electric approach, we demonstrate exosome purification with more than 95% purity and 81% recovery, well above comparable approaches.

Rapid Formation of Multicellular Spheroids in Boundary-Driven Acoustic Microstreams.

3D cell spheroid culture has emerged as a more faithful recreation of cell growth environment compared to conventional 2D culture, as it can maintain tissue structures, physicochemical characteristics, and cell phenotypes. The majority of current spheroid formation methods are limited to a physical agglomeration of the desired cell type, and then relying on cell capacity to secrete extracellular matrix to form coherent spheroids. Hence, apart from being time-consuming, their success in leading to functional spheroid formation is also cell-type dependent. In this study, a boundary-driven acoustic microstreaming tool is presented that can simultaneously congregate cells and generate sturdy cell clusters through incorporating a bioadhesive such as collagen for rapid production of spheroids. The optimized mixture of type I collagen (0.42 mg mL-1 ) and methylcellulose (0.4% w/v ) accelerates the coagulation of cell-matrix as fast as 10 s while avoiding their adhesion to the device, and thereby offering easy spheroid retrieval. The versatility of the platform is shown for the production of MDA-MB-231 and MCF-7 spheroids, multicellular spheroids, and composite spheroids made of cells and microparticles. The ability to produce densely packed spheroids embedded within a biomimetic extracellular matrix component, along with rapid formation and easy collection of spheroids render the proposed device a step in technology development required to realize potentials of 3D constructs such as building blocks for the emerging field of bottom-up tissue engineering.

Soft-Contact Acoustic Microgripper Based on a Controllable Gas-Liquid Interface for Biomicromanipulations.

The manipulation of microscale bioentities is desired in many biological and biomedical applications. However, the potential unobservable damage to bioparticles due to rigid contact has always been a source of concern. Herein, a soft-contact acoustic microgripper to handle microparticles to improve the interaction safety is introduced. The system takes advantage of the acoustic-enhanced adhesion of flexible gas-liquid interfaces to capture-release, transport, and rotate the target, such as microbeads (20-65 µm) and zebrafish embryos (from 950 µm to 1.4 mm). The gas-liquid interface generated at the tip of a microcapillary can be precisely controlled by a pneumatic pressure source. The gas-liquid interface oscillation excited by acoustic energy imposes coupled radiation force and drag force on the microparticles, enabling multidimensional movements. Experiments with the microbeads are conducted to evaluate the claimed function and quantify the key parameters that influence the manipulation result. Additionally, 250 zebrafish embryos are captured, transported, and rotated. The hatching rate of the 250 manipulated embryos is approximately 98% similar to that of the nonmanipulated group, which proves the noninvasiveness of the method. The derived theories and experimental data indicate that the developed soft-contact microgripper is functional and beneficial for biological and medical applications.

Acoustofluidic Droplet Sorter Based on Single Phase Focused Transducers.

Droplet microfluidics has revolutionized the biomedical and drug development fields by allowing for independent microenvironments to conduct drug screening at the single cell level. However, current microfluidic sorting devices suffer from drawbacks such as high voltage requirements (e.g., >200 Vpp), low biocompatibility, and/or low throughput. In this article, a single-phase focused transducer (SPFT)-based acoustofluidic chip is introduced, which outperforms many microfluidic droplet sorting devices through high energy transmission efficiency, high accuracy, and high biocompatibility. The SPFT-based sorter can be driven with an input power lower than 20 Vpp and maintain a postsorting cell viability of 93.5%. The SPFT sorter can achieve a throughput over 1000 events per second and a sorting purity up to 99.2%. The SPFT sorter is utilized here for the screening of doxorubicin cytotoxicity on cancer and noncancer cells, proving its drug screening capability. Overall, the SPFT droplet sorting device shows great potential for fast, precise, and biocompatible drug screening.

Manipulation of self-assembled three-dimensional architecture in reusable acoustofluidic device.

Reconstructing of cell architecture plays a vital role in tissue engineering. Recent developments of self-assembling of cells into three-dimensional (3D) matrix pattern using surface acoustic waves have paved a way for a better tissue engineering platform thanks to its unique properties such as nature of noninvasive and noncontact, high biocompatibility, low-power consumption, automation capability, and fast actuation. This article discloses a method to manipulate the orientation and curvature of 3D matrix pattern by redesigning the top wall of microfluidic chamber and the technique to create a 3D longitudinal pattern along preinserted polydimethylsiloxane (PDMS) rods. Experimental results showed a good agreement with model predictions. This research can actively contribute to the development of better organs-on-chips platforms with capability of controlling cell architecture and density. Meanwhile, the 3D longitudinal pattern is suitable for self-assembling of microvasculatures.

Effect of microchannel protrusion on the bulk acoustic wave-induced acoustofluidics: numerical investigation.

Acoustofluidics inside the microchannel has already found its wide applications recently. Acoustic streaming and radiation force are two underlying mechanisms that determine the trajectory of microparticles and cells in the manipulation. Critical particle size of viscous effects is found to be about 1.6 µm in the conventional rectangular microchannel (W × H = 380 m × 160 m) at the frequency of 2 MHz, below which the acoustic streaming dominants, and is independent of the driving voltage. In order to effectively adjust such a critical size, a approach is proposed and evaluated numerically to enhance the acoustic streaming by adding some protrusions (i.e., in the shape of a wedge, rod, half-ellipse) to the middle of the top or bottom wall. It is found that the resonant frequency and acoustic pressure will decrease and the acoustic streaming velocity will increase significantly, respectively, with the increase of protrusion height (up to 30 µm while keeping the width the same as 8 µm). Subsequently, trajectory motion patterns of microparticles have apparent changes in comparison to those inside the rectangular microchannel, and acoustic streaming can even dominate the motion of large microparticles (i.e., 10 µm). As a result, the critical particle size could be increased up to 72.5 µm. Furthermore, different protrusion shapes (i.e., wedge, rod, half-ellipse) on the top wall were compared. The sharpness of protrusion at its tip seems to determine the acoustic streaming velocity. The wedge attached to the bottom wall had higher resonant frequency and lower acoustic streaming velocity compared with the top wedge in the same dimension. The patterns of acoustic streaming and microparticle trajectory motion in the microchannel with dual wedges on the top and bottom walls are not the superposition of those of the top and bottom wedge individually. In summary, the geometry of the microchannel has a significant effect on the induced acoustofluidics by the bulk acoustic waves. A much larger acoustic streaming velocity is produced at the tip of the protrusion to change the critical size of microparticles between acoustic streaming and radiation force. It suggests that more applications of acoustofluidics (i.e., mixing and sonoporation) to microparticles and cells in various sizes are feasible by designing an appropriate geometry of the microchannel.

Hierarchical Nanotexturing Enables Acoustofluidics on Slippery yet Sticky, Flexible Surfaces.

The ability to actuate liquids remains a fundamental challenge in smart microsystems, such as those for soft robotics, where devices often need to conform to either natural or three-dimensional solid shapes, in various orientations. Here, we propose a hierarchical nanotexturing of piezoelectric films as active microfluidic actuators, exploiting a unique combination of both topographical and chemical properties on flexible surfaces, while also introducing design concepts of shear hydrophobicity and tensile hydrophilicity. In doing so, we create nanostructured surfaces that are, at the same time, both slippery (low in-plane pinning) and sticky (high normal-to-plane liquid adhesion). By enabling fluid transportation on such arbitrarily shaped surfaces, we demonstrate efficient fluid motions on inclined, vertical, inverted, or even flexible geometries in three dimensions. Such surfaces can also be deformed and then reformed into their original shapes, thereby paving the way for advanced microfluidic applications.