Controlling the force and the position of acoustic traps with a tunable acoustofluidic chip: Application to spheroid manipulations.

A multi-node acoustofluidic chip working on a broadband spectrum and beyond the resonance is designed for cell manipulations. A simple one-dimensional (1D) multi-layer model is used to describe the stationary standing waves generated inside a cavity. The transmissions and reflections of the acoustic wave through the different layers and interfaces lead to the creation of pressure nodes away from the resonance condition. A transparent cavity and a broadband ultrasonic transducer allow the measurement of the acoustic energy over a wide frequency range using particle image velocimetry measurements and the relation between acoustic energy and the particles velocity. The automation of the setup allows the acquisition over a large spectrum with a high frequency definition. The results show a wide continuous operating range for the acoustofluidic chip, which compares well with the 1D model. The variation of the acoustic radiation force when varying the frequency can be compensated to ensure a constant amplitude for the ARF. This approach is finally applied to mesenchymal stem cell (MCS) spheroids cultured in acoustic levitation. The MSC spheroids can be moved and merged just by varying the acoustic frequency. This approach opens the path to various acoustic manipulations and to complex 3D tissue engineering in acoustic levitation.

Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation.

In recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

Influence of the temperature on the opto-acoustophoretic effect.

Opto-acoustophoretic mobility has been demonstrated recently for fluorescent and colored particles acoustically levitated in a stationary ultrasonic field when illuminated with the appropriate optical wavelength [Dumy, Hoyos, and Aider, J. Acoust. Soc. Am. 146, 4557-4568 (2019); Zhou, Gao, Yang, Li, Shao, Zhang, Li, and Li, Adv. Sci. 5, 1800122 (2018)]. It is a repeatable phenomenon, needing both acoustic trapping and specific optic excitation to occur. However, the physical origin of the phenomenon is still debated. In this study, we provide more insights into the probable origin of this phenomenon by confronting numerical simulations with temperature controlled experiments. The phenomenon properties are well reproduced by our model, relying on a thermofluidic instability, hinting at the potential thermally induced fluid density gradient as a drag source for the observed ejection of particles. Thermostated experiments exhibit a surprising threshold above which the phenomenon is not observed anymore no matter how large the optic or acoustic energies used. This exciting observation differs from the initial interpretation of the phenomenon, altering its potential application without removing its interest because it suggests the possible contactless generation of customized flows by acoustically trapped particles.

Acoustic radiation force and torque on spheroidal particles in an ideal cylindrical chamber.

In this article, the acoustic radiation force and torque exerted on a small spheroidal particle immersed in a nonviscous fluid inside an ideal cylindrical chamber is theoretically investigated. The ideal chamber comprises a hard top and bottom (rigid boundary condition) and a soft or hard lateral wall. By assuming that the particle is much smaller than the acoustic wavelength, analytical expressions of the radiation force and torque caused by an acoustic wave of arbitrary shape are presented. Unlike previous results, these expressions are given relative to a fixed laboratory frame. The model is showcased for analyzing the behavior of an elongated metallic microspheroid (with a 10:1 aspect ratio) in a half-wavelength acoustofluidic chamber with a diameter of a few millimeters. The results show that the radiation torque aligns the microspheroid along the nodal plane, and the radiation force causes a translational motion with a speed of up to one body length per second. Finally, the implications of this study on propelled nanorods by ultrasound are discussed.

Morphological and electrical disturbances after split-flow fractionation in murine macrophages.

Split-flow fractionation (SPLITT) is a family of techniques that separates in the absence of labeling using very low flow rates and force fields, and is therefore expected to minimize cell damage. Although it has been documented that separation methods cause physiological changes in immune cells that are attributable to mechanical stress and antibody labeling, SPLITT has not yet been examined for possible damaging effects of hydrodynamic stress, partly because it is assumed that the low flow rates and weak forces used in this technique do not generate significant mechanical stress. The aim of this study was to investigate the effects of SPLITT on cell function of a murine macrophage cell, and to compare these effects with those induced by centrifugation. Macrophages J774.2 were cultured in RPMI-enriched media, then detached from the culture flask and resuspended for 12 h. Cell suspensions were diluted in a buffered saline solution and exposed to SPLITT (flow rates 1-10 ml/min) or centrifugation (100-1500g) for 10 min. Cell viability, diameter, membrane potential, and nitric oxide production were measured. Under the operating conditions employed, cell viability was above 98% after SPLITT and centrifugation but cells suffered immediate hydrodynamic cell damage, including decreased cell diameter and membrane hyperpolarization which was inhibitable by 4-aminopyridine; nitric oxide production was not affected. Pressure values during SPLITT and centrifugation correlated with diameter and membrane potential. Our data do not support the assumption that SPLITT is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Membrane potential and cell diameter are sensitive indicators for the evaluation of sublethal damage in different cell models, and allow identification of optimal operating conditions on different scales.

Observation of selective optical manipulation of particles in acoustic levitation.

Acoustic Radiation Force is commonly used to create stable large-scale aggregates of particles in levitation (so-called "acoustic levitation") in a micro-cavity. The authors show in the following work that this well-known and well-controlled aggregation process can be reversed without contact or external flow if the aggregated particles are enlightened with the proper optical wavelength. This coupled optics and acoustics effect has been observed with various kinds of particles and different optic wavelengths, showing high reproducibility. The phenomenon is studied using fluorescent micro-metric polystyrene particles without flow, and the effects of acoustic energy and illumination power have been quantitatively assessed. It is then exploited to separate a mix of particles with identical mechanical properties based on their different optic absorption. If the phenomenon is not well understood, some possible mechanisms are proposed and discussed that could be responsible for the rapid ejection of the objects in levitation from the illuminated area. Since it is a tag free phenomenon that does not need high energies to happen and since it works with biological objects such as algae, red blood cells, and bacteria, it may open the way to a broad range of applications.

Induced clustering of Escherichia coli by acoustic fields.

Brownian or self-propelled particles in aqueous suspensions can be trapped by acoustic fields generated by piezoelectric transducers usually at frequencies in the megahertz. The obtained confinement allows the study of rich collective behaviours like clustering or spreading dynamics in microgravity-like conditions. The acoustic field induces the levitation of self-propelled particles and provides secondary lateral forces to capture them at nodal planes. Here, we give a step forward in the field of confined active matter, reporting levitation experiments of bacterial suspensions of Escherichia coli. Clustering of living bacteria is monitored as a function of time, where different behaviours are clearly distinguished. Upon the removal of the acoustic signal, bacteria rapidly spread, impelled by their own swimming. Nevertheless, long periods of confinement result in irreversible bacteria entanglements that could act as seeds for levitating bacterial aggregates.

Acoustophoretic Mobility and Its Role in Optimizing Acoustofluidic Separations.

In the separation sciences, sample species are separated according to their physicochemical properties, the nature of the selective field, and, if present, the properties of the medium in which they are dissolved or suspended. Separations may be carried out on a continuous basis in microfluidic devices or split-flow thin channel (SPLITT) devices by selectively transporting species in a direction transverse to the direction of flow of the suspending fluid. Separation is achieved in the so-called transport mode according to relative differences in mobility of the species under the influence of the applied field. Gravitational, centrifugal, thermal gradient, magnetic, electric, and dielectric fields may all be used for continuous SPLITT fractionation. We present here the theory for optimizing the operation of the relatively new technique of acoustic SPLITT fractionation for the continuous separation of non-Brownian materials. The theory is based on a quantitatively defined acoustophoretic mobility that is consistent with the generalized concept of mobility proposed by Giddings. Until now, acoustophoretic mobility has almost exclusively been used as a qualitative descriptor for velocity induced by an acoustic field. The quantitative definition presented here will contribute to the advancement of all forms of acoustofluidic separations.

Acute hydrodynamic damage induced by SPLITT fractionation and centrifugation in red blood cells.

Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. Peripheral whole blood samples were collected from healthy volunteers. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1-10 ml/min) or centrifugation (100-1500 g) for 10 min. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales.

Density and Shape Effects in the Acoustic Propulsion of Bimetallic Nanorod Motors.

Bimetallic nanorods are propelled without chemical fuels in megahertz (MHz) acoustic fields, and exhibit similar behaviors to single-metal rods, including autonomous axial propulsion and organization into spinning chains. Shape asymmetry determines the direction of axial movement of bimetallic rods when there is a small difference in density between the two metals. Movement toward the concave end of these rods is inconsistent with a scattering mechanism that we proposed earlier for acoustic propulsion, but is consistent with an acoustic streaming model developed more recently by Nadal and Lauga ( Phys. Fluids 2014 , 26 , 082001 ). Longer rods were slower at constant power, and their speed was proportional to the square of the power density, in agreement with the acoustic streaming model. The streaming model was further supported by a correlation between the disassembly of spinning chains of rods and a sharp decrease in the axial speed of autonomously moving motors within the levitation plane of the cylindrical acoustic cell. However, with bimetallic rods containing metals of different densities, a consistent polarity of motion was observed with the lighter metal end leading. Speed comparisons between single-metal rods of different densities showed that those of lower density are propelled faster. So far, these density effects are not explained in the streaming model. The directionality of bimetallic rods in acoustic fields is intriguing and offers some new possibilities for designing motors in which shape, material, and chemical asymmetry might be combined for enhanced functionality.

Study of the onset of the acoustic streaming in parallel plate resonators with pulse ultrasound.

In a previous study, we introduced pulse mode ultrasound as a new method for reducing and controlling the acoustic streaming in parallel plate resonators (Hoyos and Castro, 2013). Here, by modifying other parameters such as the resonator geometry and the particle size, we have found a threshold for particle manipulation with ultrasonic standing waves in confined resonators without the influence of the acoustic streaming. We demonstrate that pulse mode ultrasound open the possibility of manipulating particles smaller than 1 µm size.

Experimental study on inter-particle acoustic forces.

A method for the experimental measurement of inter-particle forces (secondary Bjerknes force) generated by the action of an acoustic field in a resonator micro-channel is presented. The acoustic radiation force created by an ultrasonic standing wave moves suspended particles towards the pressure nodes and the acoustic pressure induces particle volume oscillations. Once particles are in the levitation plane, transverse and secondary Bjerknes forces become important. Experiments were carried out in a resonator filled with a suspension composed of water and latex particles of different size (5-15 µm) at different concentrations. Ultrasound was generated by means of a 2.5 MHz nominal frequency transducer. For the first time the acoustic force generated by oscillating particles acting on other particles has been measured, and the critical interaction distance in various cases has been determined. Inter-particle forces on the order of 10(-14) N have been measured by using this method.

Steering acoustically propelled nanowire motors toward cells in a biologically compatible environment using magnetic fields.

The recent discovery of fuel-free propulsion of nanomotors using acoustic energy has provided a new avenue for using nanomotors in biocompatible media. Crucial to the application of nanomotors in biosensing and biomedical applications is the ability to remotely control and steer them toward targets of interest, such as specific cells and tissues. We demonstrate in vitro magnetic steering of acoustically powered nanorod motors in a biologically compatible environment. Steering was accomplished by incorporating (40 ± 5) nm thick nickel stripes into the electrochemically grown nanowires. An external magnetic field of 40-45 mT was used to orient the motors, which were acoustically propelled along their long axes. In the absence of a magnetic field, (300 ± 30) nm diameter, (4.3 ± 0.2) µm long nanowires with (40 ± 5) nm thick magnetic stripes exhibit the same self-acoustophoretic behavior, including pattern formation into concentric nanowire circles, aligned spinning chains, and autonomous axial motion, as their non-magnetic counterparts. In a magnetic field, these wires and their paths are oriented as evidenced by their relatively linear trajectories. Coordinated motion of multiple motors and targeting of individual motors toward HeLa cells with micrometer-level precision was demonstrated.

Controlling the acoustic streaming by pulsed ultrasounds.

We propose a technique based on pulsed ultrasounds for controlling, reducing to a minimum observable value the acoustic streaming in closed ultrasonic standing wave fluidic resonators. By modifying the number of pulses and the repetition time it is possible to reduce the velocity of the acoustic streaming with respect to the velocity generated by the continuous ultrasound mode of operation. The acoustic streaming is observed at the nodal plane where a suspension of 800nm latex particles was focused by primary radiation force. A mixture of 800nm and 15µm latex particles has been also used for showing that the acoustic streaming is hardly reduced while primary and secondary forces continue to operate. The parameter we call "pulse mode factor" i.e. the time of applied ultrasound divided by the duty cycle, is found to be the adequate parameter that controls the acoustic streaming. We demonstrate that pulsed ultrasound is more efficient for controlling the acoustic streaming than the variation of the amplitude of the standing waves.

Autonomous motion of metallic microrods propelled by ultrasound.

Autonomously moving micro-objects, or micromotors, have attracted the attention of the scientific community over the past decade, but the incompatibility of phoretic motors with solutions of high ionic strength and the use of toxic fuels have limited their applications in biologically relevant media. In this letter we demonstrate that ultrasonic standing waves in the MHz frequency range can levitate, propel, rotate, align, and assemble metallic microrods (2 µm long and 330 nm diameter) in water as well as in solutions of high ionic strength. Metallic rods levitated to the midpoint plane of a cylindrical cell when the ultrasonic frequency was tuned to create a vertical standing wave. Fast axial motion of metallic microrods at ~200 µm/s was observed at the resonant frequency using continuous or pulsed ultrasound. Segmented metal rods (AuRu or AuPt) were propelled unidirectionally with one end (Ru or Pt, respectively) consistently forward. A self-acoustophoresis mechanism based on the shape asymmetry of the metallic rods is proposed to explain this axial propulsion. Metallic rods also aligned and self-assembled into long spinning chains, which in the case of bimetallic rods had a head-to-tail alternating structure. These chains formed ring or streak patterns in the levitation plane. The diameter or distance between streaks was roughly half the wavelength of the ultrasonic excitation. The ultrasonically driven movement of metallic rods was insensitive to the addition of salt to the solution, opening the possibility of driving and controlling metallic micromotors in biologically relevant media using ultrasound.

Controlled cell aggregation in a pulsed acoustic field.

Cell aggregation in ultrasonic resonators can be obtained in a few seconds. Hundreds even thousands of cells can be levitated in suspension and generate 2D or 3D aggregates. Nevertheless, the aggregation rate and the 2D or 3D configurations of the resultant aggregates are very difficult to control. This work reports on a novel way of generating and controlling particle and cell aggregates using pulsed ultrasound. This technique specifically explores (in addition to the ultrasound wave, frequency and amplitude) the time of ultrasound application, i.e. the number of pulses as well as the pulse repetition frequency. We demonstrate that with pulsed ultrasound, particles and/or cells levitate in suspension, as with continuous ultrasound, and the aggregation rate can be modified in a controlled manner. By carefully tuning the number of pulses and the repetition frequency, the 3-D and 2-D configurations of the aggregates can be selectively generated. In addition, pulsed ultrasound limits transducer heating, thus allowing for higher acoustic energies than those currently employed with continuous ultrasound.

The use of a linear Halbach array combined with a step-SPLITT channel for continuous sorting of magnetic species.

The Quadrupole Magnetic Sorter (QMS), employing an annular flow channel concentric with the aperture of a quadrupole magnet, is well established for cell and particle separations. Here we propose a magnetic particle separator comprising a linear array of cylindrical magnets, analogous to the array proposed by Klaus Halbach, mated to a substantially improved form of parallel-plate SPLITT channel, known as the step-SPLITT channel. While the magnetic force and throughput are generally lower than for the QMS, the new separator has advantages in ease of fabrication and the ability to vary the magnetic force to suit the separands. Preliminary experiments yield results consistent with prediction and show promise regarding future separations of cells of biomedical interest.

Enhanced diffusion due to active swimmers at a solid surface.

We consider two systems of active swimmers moving close to a solid surface, one being a living population of wild-type E. coli and the other being an assembly of self-propelled Au-Pt rods. In both situations, we have identified two different types of motion at the surface and evaluated the fraction of the population that displayed ballistic trajectories (active swimmers) with respect to those showing randomlike behavior. We studied the effect of this complex swimming activity on the diffusivity of passive tracers also present at the surface. We found that the tracer diffusivity is enhanced with respect to standard Brownian motion and increases linearly with the activity of the fluid, defined as the product of the fraction of active swimmers and their mean velocity. This result can be understood in terms of series of elementary encounters between the active swimmers and the tracers.

On the no-field method for void time determination in flow field-flow fractionation.

Elution time measurements of colloidal particles injected in a symmetrical flow field-flow fractionation (flow FFF) system when the inlet and outlet cross-flow connections are closed have been performed. This no-field method has been proposed earlier for void time (and void volume) determination in flow FFF Giddings et al. (1977). The elution times observed were much larger than expected on the basis of the channel geometrical volume and the flow rate. In order to explain these discrepancies, a flow model allowing the carrier liquid to flow through the porous walls toward the reservoirs located behind the porous elements and along these reservoirs was developed. The ratio between the observed elution time and expected one is found to depend only on a parameter which is a function of the effective permeability and thickness of the porous elements and of the channel thickness and length. The permeabilities of the frits used in the system were measured. Their values lead to predicted elution times in reasonable agreement with experimental ones, taking into account likely membrane protrusion inside the channel on system assembly. They comfort the basic feature of the flow model, in the no-field case. The carrier liquid mostly bypasses the channel to flow along the system mainly in the reservoir. It flows through the porous walls toward the reservoirs near channel inlet and again through the porous walls from the reservoirs to the channel near channel outlet before exiting the system. In order to estimate the extent of this bypassing process, it is desirable that the hydrodynamic characteristics of the permeable elements (permeability and thickness) are provided by flow FFF manufacturers. The model applies to symmetrical as well as asymmetrical flow FFF systems.