Hydrodynamic coupling melts acoustically levitated crystalline rafts.

Going beyond the manipulation of individual particles, first steps have recently been undertaken with acoustic levitation in air to investigate the collective dynamical properties of many-body systems self-assembled within the levitation plane. However, these assemblies have been limited to two-dimensional, close-packed rafts where forces due to scattered sound pull particles into direct frictional contact. Here, we overcome this restriction using particles small enough that the viscosity of air establishes a repulsive streaming flow at close range. By tuning the particle size relative to the characteristic length scale for viscous streaming, we control the interplay between attractive and repulsive forces and show how particles can be assembled into monolayer lattices with tunable spacing. While the strength of the levitating sound field does not affect the particles' steady-state separation, it controls the emergence of spontaneous excitations that can drive particle rearrangements in an effectively dissipationless, underdamped environment. Under the action of these excitations, a quiescent particle lattice transitions from a predominantly crystalline structure to a two-dimensional liquid-like state. We find that this transition is characterized by dynamic heterogeneity and intermittency, involving cooperative particle movements that remove the timescale associated with caging for the crystalline lattice. These results shed light on the nature of athermal excitations and instabilities that can arise from strong hydrodynamic coupling among interacting particles.

Acoustic manipulation of multi-body structures and dynamics.

Sound can exert forces on objects of any material and shape. This has made the contactless manipulation of objects by intense ultrasound a fascinating area of research with wide-ranging applications. While much is understood for acoustic forcing of individual objects, sound-mediated interactions among multiple objects at close range gives rise to a rich set of structures and dynamics that are less explored and have been emerging as a frontier for research. We introduce the basic mechanisms giving rise to sound-mediated interactions among rigid as well as deformable particles, focusing on the regime where the particles' size and spacing are much smaller than the sound wavelength. The interplay of secondary acoustic scattering, Bjerknes forces, and micro-streaming is discussed and the role of particle shape is highlighted. Furthermore, we present recent advances in characterizing non-conservative and non-pairwise additive contributions to the particle interactions, along with instabilities and active fluctuations. These excitations emerge at sufficiently strong sound energy density and can act as an effective temperature in otherwise athermal systems.

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.

Acoustic Levitation Synthesis of Ultrahigh-Density Spherical Nucleic Acid Architectures for Specific SERS Analysis.

Controllably regulating the electrostatic bilayer of nanogold colloids is a significant premise for synthesizing spherical nucleic acid (SNA) and building ordered plasmonic architectures. We develop a facile acoustic levitation reactor to universally synthesize SNAs with an ultra-high density of DNA strands, which is even higher than those of various state-of-the-art methods. Results reveal a new mechanism of DNA grafting via acoustic wave that can reconfigure the ligands on colloidal surfaces. The acoustic levitation reactor enables substrate-free three-dimentional (3D) spatial assembly of SNAs with controllable interparticle nanogaps through regulating DNA lengths. This kind of architecture may overcome the plasmonic enhancement limits by blocking electron tunneling and breaking electrostatic shielding in dried aggregations. Finite element simulations support the architecture with 3D spatial plasmonic hotspot matrix, and its ultrahigh surface-enhanced Raman scattering (SERS) capability is evidenced by in situ untargeted tracking of biomolecular events during photothermal stimulation (PTS)-induced cell death process. For biomarker diagnosis, the conjugation of adenosine triphosphate (ATP) aptamer onto SNAs enables in situ targeted tracking of ATP during PTS-induced cell death process. Particularly, the CD71 receptor and integrin α3ß1 protein on PL45 cell membrance could be well distinguished by label-free SERS fingerprints when using specific XQ-2d and DML-7 aptamers, respectively, to synthesize SNA architectures. Our current acoustic levitation reactor offers a new method for synthesizing SNAs and enables both targeted and untargeted SERS analysis for tracking molecular events in living systems. It promises great potentials in biochemical synthesis and sensing in future.

Holographic acoustic tweezers.

Acoustic tweezers use sound radiation forces to manipulate matter without contact. They provide unique characteristics compared with the more established optical tweezers, such as higher trapping forces per unit input power and the ability to manipulate objects from the micrometer to the centimeter scale. They also enable the trapping of a wide range of sample materials in various media. A dramatic advancement in optical tweezers was the development of holographic optical tweezers (HOT) which enabled the independent manipulation of multiple particles leading to applications such as the assembly of 3D microstructures and the probing of soft matter. Now, 20 years after the development of HOT, we present the realization of holographic acoustic tweezers (HAT). We experimentally demonstrate a 40-kHz airborne HAT system implemented using two 256-emitter phased arrays and manipulate individually up to 25 millimetric particles simultaneously. We show that the maximum trapping forces are achieved once the emitting array satisfies Nyquist sampling and an emission phase discretization below π/8 radians. When considered on the scale of a wavelength, HAT provides similar manipulation capabilities as HOT while retaining its unique characteristics. The examples shown here suggest the future use of HAT for novel forms of displays in which the objects are made of physical levitating voxels, assembly processes in the micrometer and millimetric scale, as well as positioning and orientation of multiple objects which could lead to biomedical applications.

Measurement of Secondary Structure Changes in Poly-L-lysine and Lysozyme during Acoustically Levitated Single Droplet Drying Experiments by In Situ Raman Spectroscopy.

Drying processes such as spray drying, as commonly used in the pharmaceutical industry to convert protein-based drugs into their particulate form, can lead to an irreversible loss of protein activity caused by protein secondary structure changes. Due to the nature of these processes (high droplet number, short drying time), an in situ investigation of the structural changes occurring during a real drying process is hardly possible. Therefore, an approach for the in situ investigation of the expected secondary structural changes during single droplet protein drying in an acoustic levitator by time-resolved Raman spectroscopy was developed and is demonstrated in this paper. For that purpose, a self-developed NIR-Raman sensor generates and detects the Raman signal from the levitated solution droplet. A mathematical spectral reconstruction by multiple Voigt functions is used to quantify the relative secondary structure changes occurring during the drying process. With the developed setup, it was possible to detect and quantify the relative secondary structure changes occurring during single droplet drying experiments for the two chosen model substances: poly-L-lysine, a homopolypeptide widely used as a protein mimic, and lysozyme. Throughout drying, an increase in the ß-sheet structure and a decrease in the other two structural elements, α-helix, and random coil, could be identified. In addition, it was observed that the degree of structural changes increased with increasing temperature.

Insights into the evaporation characteristics of saliva droplets and aerosols: Levitation experiments and numerical modeling.

Understanding the transmission phenomena of SARS-CoV-2 by virus-laden droplets and aerosols is of paramount importance for controlling the current COVID-19 pandemic. Detailed information about the lifetime and kinematics of airborne droplets of different size is relevant in order to evaluate hygiene measures like wearing masks but also social distancing and ventilation concepts for indoor environments. However, the evaporation process of expiratory droplets and aerosols is not fully understood. Consequently, the main objective of this study is to present evaporation characteristics of saliva droplets. An acoustic levitator is utilized in conjunction with microscopic imaging for recording the temporal evolution of the evaporation of saliva droplets under well-defined ambient conditions. Following the evaporation of the water content, a saliva droplet reaches a final size, which remains stable in the timescale of hours. By investigating numerous droplets of different size, it was found that the final droplet diameter correlates well to 20 % of the initial diameter. This correlation is independent of the ambient conditions for a temperature range from 20  °C to 29  °C and a relative humidity from 6 % to up to 65 %. The experimentally obtained evaporation characteristics are implemented into a numerical model, which is based on one-dimensional droplet kinematics and a rapid mixing evaporation model. By taking into account the evaporation-falling curve as presented by Wells, the significance of the experimental results for predicting the lifetime of saliva droplets and aerosols is demonstrated. The numerical predictions may be used to determine the impact of the droplet size and the ambient conditions on the transmission risks of infectious diseases like COVID-19.

Modeling Drying Behavior of an Aqueous Chitosan Single Droplet Using the Reaction Engineering Approach.

Spray drying of Chitosan solutions to prepare microparticles either using pilot or industrial scale spray dryer is a complex process; tracking morphological changes and obtaining drying kinetics of a single droplet would be very difficult. The acoustic levitator being a non-intrusive method is a useful experimental apparatus that enables particle/droplet suspension in the gaseous medium and capable of mimicking the drying process in a spray dryer. The drying of chitosan aqueous solutions into solid particles was investigated. The prediction of the size and drying kinetics until the formation of the solid structure was performed in an acoustic levitator. Studying the drying of single droplets is crucial for revealing the influence of the drying process parameters on the formation of dried particles. Droplets with initial chitosan concentration (10, 20, and 30 mg/ml) were investigated at different air-drying temperatures. A Reaction Engineering Approach (REA) model was developed and compared with the experimental drying curves, a very well agreement was found between the drying experiments and the REA model with a relative error of about 3% between the initial droplet mass and predicted droplet mass by the REA model.

Laser ionization ion mobility spectrometric interrogation of acoustically levitated droplets.

Acoustically levitated droplets have been suggested as compartmentalized, yet wall-less microreactors for high-throughput reaction optimization purposes. The absence of walls is envisioned to simplify up-scaling of the optimized reaction conditions found in the microliter volumes. A consequent pursuance of high-throughput chemistry calls for a fast, robust and sensitive analysis suited for online interrogation. For reaction optimization, targeted analysis with relatively low sensitivity suffices, while a fast, robust and automated sampling is paramount. To follow this approach, in this contribution, a direct coupling of levitated droplets to a homebuilt ion mobility spectrometer (IMS) is presented. The sampling, transfer to the gas phase, as well as the ionization are all performed by a single exposure of the sampling volume to the resonant output of a mid-IR laser. Once formed, the nascent spatially and temporally evolving analyte ion cloud needs to be guided out of the acoustically confined trap into the inlet of the ion mobility spectrometer. Since the IMS is operated at ambient pressure, no fluid dynamic along a pressure gradient can be employed. Instead, the transfer is achieved by the electrostatic potential gradient inside a dual ring electrode ion optics, guiding the analyte ion cloud into the first stage of the IMS linear drift tube accelerator. The design of the appropriate atmospheric pressure ion optics is based on the original vacuum ion optics design of Wiley and McLaren. The obtained experimental results nicely coincide with ion trajectory calculations based on a collisional model. Graphical Abstract.

Mass Spectrometric Study of Acoustically Levitated Droplets Illuminates Molecular-Level Mechanism of Photodynamic Therapy for Cancer involving Lipid Oxidation.

Even though the general mechanism of photodynamic cancer therapy is known, the details and consequences of the reactions between the photosensitizer-generated singlet oxygen and substrate molecules remain elusive at the molecular level. Using temoporfin as the photosensitizer, here we combine field-induced droplet ionization mass spectrometry and acoustic levitation techniques to study the "wall-less" oxidation reactions of 18:1 cardiolipin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) mediated by singlet oxygen at the air-water interface of levitated water droplets. For both cardiolipin and POPG, every unsaturated oleyl chain is oxidized to an allyl hydroperoxide, which surprisingly is immune to further oxidation. This is attributed to the increased hydrophilicity of the oxidized chain, which attracts it toward the water phase, thereby increasing membrane permeability and eventually triggering cell death.

Using containerless methods to develop amorphous pharmaceuticals.

BACKGROUND: Many pipeline drugs have low solubility in their crystalline state and require compounding in special dosage forms to increase bioavailability for oral administration. The use of amorphous formulations increases solubility and uptake of active pharmaceutical ingredients. These forms are rapidly gaining commercial importance for both pre-clinical and clinical use. METHODS: Synthesis of amorphous drugs was performed using an acoustic levitation containerless processing method and spray drying. The structure of the products was investigated using in-situ high energy X-ray diffraction. Selected solvents for processing drugs were investigated using acoustic levitation. The stability of amorphous samples was measured using X-ray diffraction. Samples processed using both spray drying and containerless synthesis were compared. RESULTS: We review methods for making amorphous pharmaceuticals and present data on materials made by containerless processing and spray drying. It was shown that containerless processing using acoustic levitation can be used to make phase-pure forms of drugs that are known to be difficult to amorphize. The stability and structure of the materials was investigated in the context of developing and making clinically useful formulations. CONCLUSIONS: Amorphous compounds are emerging as an important component of drug development and for the oral delivery of drugs with low solubility. Containerless techniques can be used to efficiently synthesize small quantities of pure amorphous forms that are potentially useful in pre-clinical trials and for use in the optimization of clinical products. GENERAL SIGNIFICANCE: Developing new pharmaceutical products is an essential enterprise to improve patient outcomes. The development and application of amorphous pharmaceuticals to increase absorption is rapidly gaining importance and it provides opportunities for breakthrough research on new drugs. There is an urgent need to solve problems associated with making formulations that are both stable and that provide high bioavailability. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Combination of acoustic levitation with small angle scattering techniques and synchrotron radiation circular dichroism. Application to the study of protein solutions.

BACKGROUND: The acoustic levitation technique is a useful sample handling method for small solid and liquids samples, suspended in air by means of an ultrasonic field. This method was previously used at synchrotron sources for studying pharmaceutical liquids and protein solutions using x-ray diffraction and small angle x-ray scattering (SAXS). METHODS: In this work we combined for the first time this containerless method with small angle neutron scattering (SANS) and synchrotron radiation circular dichroism (SRCD) to study the structural behavior of proteins in solutions during the water evaporation. SANS results are also compared with SAXS experiments. RESULTS: The aggregation behavior of 45µl droplets of lysozyme protein diluted in water was followed during the continuous increase of the sample concentration by evaporating the solvent. The evaporation kinetics was followed at different drying stage by SANS and SAXS with a good data quality. In a prospective work using SRCD, we also studied the evolution of the secondary structure of the myoglobin protein in water solution in the same evaporation conditions. CONCLUSIONS: Acoustic levitation was applied for the first time with SANS and the high performances of the used neutron instruments made it possible to monitor fast container-less reactions in situ. A preliminary work using SRCD shows the potentiality of its combination with acoustic levitation for studying the evolution of the protein structure with time. GENERAL SIGNIFICANCE: This multi-techniques approach could give novel insights into crystallization and self-assembly phenomena of biological compound with promising potential applications in pharmaceutical, food and cosmetics industry. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Following in Real Time the Two-Step Assembly of Nanoparticles into Mesocrystals in Levitating Drops.

Mesocrystals composed of crystallographically aligned nanocrystals are present in biominerals and assembled materials which show strongly directional properties of importance for mechanical protection and functional devices. Mesocrystals are commonly formed by complex biomineralization processes and can also be generated by assembly of anisotropic nanocrystals. Here, we follow the evaporation-induced assembly of maghemite nanocubes into mesocrystals in real time in levitating drops. Analysis of time-resolved small-angle X-ray scattering data and ex situ scanning electron microscopy together with interparticle potential calculations show that the substrate-free, particle-mediated crystallization process proceeds in two stages involving the formation and rapid transformation of a dense, structurally disordered phase into ordered mesocrystals. Controlling and tailoring the particle-mediated formation of mesocrystals could be utilized to assemble designed nanoparticles into new materials with unique functions.

The resonant behavior of airborne standing-wave acoustic levitators based on arrays of ultrasonic transducers.

Recently airborne standing-wave acoustic levitation has seen great advances, and its applicability has been broadened due to the development of cavities constructed with arrays of compact ultrasonic sources. Yet, the numerical methods employed to study and predict the pressure distributions inside these cavities do not consider the effect of multiple reflections on the boundaries, hiding their resonant effects. This work presents an analytical, numerical, and experimental study of the effect of multiple reflections inside ultrasonic cavities based on arrays of transducers exhibiting their influence on the pressure amplitudes of focused standing waves. Our numerical results come from a modified version of the Matrix Method to numerically compute the multiple wave reflections of cavities constructed by two opposite arrays of multiple compact sources as boundaries. The correlation between numerical and experimental results reveals that intra-cavity reflections are relevant in focused axisymmetric cavities based on two arrays of multiple ultrasonic sources having a considerable impact on the amplitude of the standing waves and consequently, on the acoustic levitation performance. Thus, intra-cavity reflections must be considered for optimal cavity designs.

Improved functionality of hepatic spheroids cultured in acoustic levitation compared to existing 2D and 3D models.

Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture.

Drop Dissolution Intensified by Acoustic Levitation.

Acoustic levitation can provide significant benefits for many fundamental research questions. However, it is important to consider that the acoustic field influences the measurement environment. This work focuses on the dissolution of immobilised drops using acoustic levitation in liquid-liquid systems. Previous work demonstrated that the acoustic field of standing waves impacts mass transfer by affecting the spread of dissolved substances in the continuous phase in two distinct ways: (I) solutes may either pass through nodal planes of the standing waves or (II) not pass. The binary systems examined for case (I) are 1-hexanol-water and 1-butanol-water, and for case (II), n-butyl acetate-water and toluene-water. This work quantifies the intensification effect of acoustic levitation on dissolution for the two types of behaviour, by comparing them with reference measurements of mechanically attached dissolving drops. The system was designed to ensure minimal intensification. The minimum intensification of mass transfer for levitating drops in the used setup of case (I) was 25%, and for case (II), it was 65%, both increasing with decreasing surface-equivalent diameter. With this understanding, acoustic levitation can be used more accurately in the field of mass transfer studies.

Enhanced standing-wave acoustic levitation using high-order transverse modes in phased array ultrasonic cavities.

Airborne acoustic trapping by ultrasonic phased arrays has seen great advances in recent years, and yet the manipulation of objects with different shapes and sizes or heavy particles remains challenging. Here, we demonstrate that the manipulation capabilities of a standing-wave acoustic levitator can be extended by introducing intracavity high-order transverse (HOT) modes in the azimuthal direction, enabling the simultaneous trapping of several objects within a wide range of shapes and sizes with positional and rotational stability, including objects with sizes larger than one wavelength and weights in the scale of millinewtons. The conditions to generate different HOT modes are theoretically analyzed and experimentally implemented. We numerically calculate the pressure distributions, exhibiting good qualitative agreement with the experimental pressure distributions obtained with schlieren images. In addition, we calculate the acoustic force field for several examples of HOT modes and different particle sizes, which leads to a qualitative understanding of the experimental observations.

CO2 induced phase transition on a self-standing droplet studied by X-ray scattering and magnetic resonance.

HYPOTHESIS: Acoustic levitation is a suitable approach for studying processes occurring at the gas-liquid interfaces, as it allows its investigation in a contact-free manner while providing control over the gas phase. Here, we hypothesize that phase transitions induced by a CO2 rich atmosphere can be examined, at different length scales, in a contact-free manner. EXPERIMENTAL: A system consisting of 12-hydroxysteric acid (HSA) soaps mixed with different ratios of monoethanolamine (MEA) and choline hydroxide, was prepared. Microliter droplets of the samples were acoustically levitated and monitored with a camera, while exposed to CO2 to modify the pH through diffusion at the air-liquid interface and inside the droplet. The phase transition and water mobility in the levitated droplets were evaluated through X-ray scattering (SAXS/WAXS) and magnetic resonance studies, in real-time. Finally, the droplets were collected and examined under the microscope. FINDINGS: The introduction of CO2 gas induced a phase transition from micelles to multi-lamellar tubes, resulting in a gel-like behavior both in the bulk and at the interface. The high stability of the acoustic levitator allowed the investigation of this dynamic phenomenon, in real-time, in a contact-free environment. This study showcases the suitability of acoustic levitation as a tool to investigate complex chemical processes at interfaces.

Contactless ultrasound droplet manipulation system for mixing chemical reagents.

Green chemistry has been a rising topic in environmental sustainability, with a focus on the waste and consumption reduction of chemical and biomedical industries. Traditional chemical handling processes require tools that contact chemical reagents to produce vast amounts of residues and disposals. This study presents a contactless chemical mixing system that integrates acoustic droplet ejection and levitation techniques. First, the acoustic droplet ejection system creates a droplet in mid-air from a designated liquid reservoir by focusing acoustic energy at the liquid-air junction. The droplet levitation system captures and transports the droplet along a predetermined path by shifting the focal points of the acoustic standing waves. This facilitates contactless mixing of chemicals in a defined ratio. Notably, this study employs piezoelectric discs in an acoustic droplet ejection system to eject droplets from liquids. The relationship between the duration of the driving bursts and height and size of ejected droplets was also investigated. The proposed acoustic standing wave levitation system captures droplets with weights between 2.8 and 5.2 mg. To assess the reliability of the proposed system, 25 droplets were sequentially generated and transported to the mixing well without failure. The root mean square error between the collected and expected liquid weights was only 0.098 mg. The proposed system offers a promising solution for reducing waste and promoting environmentally friendly practices in chemical and biomedical laboratories.

Analysis of dynamic levitation process of the particle chain in a nonlinear standing wave field.

This research delves into the dynamic behavior of acoustic levitation of the particle chain in a nonlinear standing wave field. Experimental acoustic levitation control tests reveal bifurcation and jump phenomena during dynamic adjustments to resonant cavity height. Employing the 10-particle chain experiments and the COMSOL simulation models, the Sine-Gordon 2D vibration model is established to study the dynamic deformation process of the particle chain. The study uncovers the nonlinear interaction of particle lateral vibrations, horizontal acoustic radiation force, and conical wave fields that generate the jumping standing wave field. Notably, the fourth particle acts as a prominent jumping critical point in the secondary standing wave field, facilitating the derivation of the particle chain's nonlinear levitation dynamics. This discovery provides us with a new method to regulate the particle chain system.